Leaves blow in the wind as the camera pans through trees as the movie title is shown. The camera continues going downward, until the House of Mouse is seen.
Pooh, Piglet, Tigger, Bambi, the Hag, Pete, Big Bad Wolf, Hades and several others walk down the road, and are let inside by Max. He watches them enter as the sound of a thunder crash is head. Max turns to see the Cave of Wonders has appeared in front of the club, having seemingly burst from the ground. Jafar exits and leaves the golden scarab (which is supposed to make up the Cave's eyes) with Max, wishing him a happy Halloween. Max is left dumbfounded as he eyes the scarab as a car alarm chirps.
Inside the lobby, Donald looks in the Magic Mirror, admiring his devil costume; as it's Halloween, he's hoping to scare someone. Daisy overhears him and tells him on the "scream-o-meter" he's barely a squeal. Goofy then tells Donald that Daisy is right; he's not going to scare someone dressed up as a big red bunny (as Goofy mistakenly believes). He then tries all night to scare someone, as a side gag.
All of Donald's attempts backfire on him; (these happen at separate times after a cartoon is shown) he tries scaring Beast (very bad idea) and gets roared at, getting scared himself. He next tries to sneak up on the Fates. However, the short one points their eye at him, showing they know what he's up to. Donald's last attempt is to scare the Queen, who changes into her Hag form and scares him. Donald is left depressed, as he cannot scare someone.
Backstage, Minnie takes notice to the large group of villains in the audience (Chernabog, Professor Ratigan, Claude Frollo, Lady Tremaine, the Wicked Queen and Maleficent specifically) and tries to make Mickey worry that they may be up to something. However, Mickey assures Minnie that they have so many villains as guests at the club tonight because it's Halloween; he believes they won't be up to any tricks.
While waiting for Mickey to start the Halloween festivities, Cruella De Vil complains to her fellow villains every Halloween at the House of Mouse only has treats and no tricks, claiming that if she ran it, things would be different. Ursula asks if she'd add a "splash" of evil while Captain Hook thinks it would be pillage and plundering. Iago complains that all the villains do is talk, thus making them dull. Jafar, however, decides to end that and tells them that he has "a trick for Mickey Mouse", but they will have to wait until midnight; his cohorts are enticed by this and now eagerly await his plot's unfolding. Mic introduces Mickey (in a vampire costume), who tells the crowd some jokes before starting the cartoons.
After five cartoons, Iago wonders how much longer it will be until Jafar's trick, which Jafar assures him it will be after the next cartoon, following which, the five of them sneak backstage. While Mickey is taking bows onstage, Ursula grabs Minnie to keep her quiet. Jafar apologizes, but explains they don't want to be dull villains on Halloween. He then cues Hook, who cues Cruella to turn off the power ("Lights out!") as Mickey announces to the crowd that it is time for a change of pace.
Jafar then appears on stage telling him that he's right and begins a song. Cruella frees some evil ghosts from a box labeled "Hitchhiking Ghosts" in the prop room and they transform the club into a darkened, evil version of itself, with mist floating on the floor. The other villains, seeing this as a chance to have their own exclusive club, join in singing It's Our House Now! Hitchhiking Ghosts sing "Lock the doors!" while scaring Winnie-the-Pooh & Piglet and All The Villains force all the non-villainous characters into the kitchen and the Queen of Hearts makes her card soldiers keeping an eye on them. They next kick Mickey and the rest of the House of Mouse staff out and then all Villains spin the club's sign changes to say "House of Villains". After which, the outside changes to a blue ground floor with the top half being completely crimson. But the rest of the House of Mouse staff did not get kicked out. They hid from all of the Villains inside the House of Villains.
Taking the stage, Jafar announces to the villains that since the House of Mouse is now theirs, things will be run differently. However, Mickey returns, telling Jafar that he's had his fun and better turns things back to normal; he's even brought his boys (Donald and Goofy) as back up. Jafar is then backed up by Hades, Ursula and Chernabog. Intimidated by the large demon, Mickey and his friends are thrown out as Jafar starts the next cartoon, calling "especially that scaredy duck Donald" are about to get a real fright. (The cartoon has Donald steal Halloween candy from his nephews who get revenge on him; Goofy also gets his holidays mixed up, thinking it's either Easter or Christmas)
After the cartoon, Mickey and his friends look inside, wondering what they can do. Minnie, fed up with the villains, marches inside. Goofy says he's never seen Minnie so mad, but Mickey tells him he has. Jafar announces to the crowd that he has saved the best cartoon for last just as Minnie begins yelling at him; she tells him he and the other villains have 30 seconds to change everything back to normal. As Minnie counts down, Jafar motions for help and Captain Hook picks Minnie up and tosses her out as Jafar makes a pun about the next cartoon being a real scream; Minnie screams as he says this. The cartoon shown is a Mickey and Minnie version of "Hansel and Gretel". At the end of the cartoon, Mickey and his friends sneak backstage and dress him in his sorcerer robes from Fantasia. The villains become surprised by Mickey's reappearance as a sorcerer; Jafar fires a blast at Mickey, who grabs the fire and turns it into a ball that he tosses back at Jafar; the ball is hit backstage, where Donald back evades it. Mickey conjures a ball and throws it at Jafar, who misses it; the ball takes off Hook's hat. Mickey sends another ball at Jafar, who bunts it off his staff. The fire ball bounces back at Mickey, whose magic hat is knocked off; he has no time to put it back on. Jafar moves in to eliminate the heroic mouse. However, all the Villains are watching Jafar and Mickey, Aladdin, who escaped the kitchen and into the prop basement, opens the backdoor and tosses a magic lamp to Mickey. The other villains flee in terror, realizing that their leader has been defeated while Mickey sucks Jafar into the lamp, all the guests from the kitchen are now free. Everyone cheering as Tinker Bell flies to the club's sign saying "House of Villains", and uses her magic to change the sign back to "House of Mouse". Everyone cheers for Mickey and as Minnie gives Mickey a kiss to thank him, telling him he's the only real leader of the club. Donald pops up behind them in a Jafar costume, but Mickey tells him that even though he finally picked a scary costume, Goofy beat him to it; he's dressed as Jafar, too. Donald ends the movie with his classic line "Oh phooey!"
- Wayne Allwine as Mickey Mouse
- Russi Taylor as Minnie Mouse, Grandma Duck
- Bill Farmer as Goofy
- Tony Anselmo as Donald Duck
- Tress MacNeille as Daisy Duck, Queen of Hearts, The Fates, Si and Am, Anastasia
- Jonathan Freeman as Jafar
- Gilbert Gottfried as Iago
- Corey Burton as Captain Hook, Chernabog
- Matt Frewer as Panic
- Bobcat Goldthwait as Pain
- Jeff Bennett as Pirate Crew
- James Woods as Hades
- Susanne Blakeslee as Cruella De Vil, Madame Medusa
- Bob Joles as Mr. Snoops
- Pat Carroll as Ursula
- Rob Paulsen as Hades (singing voice)
- Jim Cummings as Pete, Big Bad Wolf, Kaa, Ed
- Lois Nettleton as Maleficent
- Scott Weinger as Aladdin
- Robby Benson as Beast (uncredited)
- Rod Roddy as Mike the Microphone
- John Fiedler as Piglet
Disney Villains by movie/TV show
- Classic Disney Cartoons and Comic Books: Pete, Lonesome Ghosts and Big Bad Wolf
- Snow White and the Seven Dwarfs: The Evil Queen (both normal and Witch form)
- Pinocchio: Honest John and Gideon (unseen) and Stromboli
- Fantasia: Chernabog
- The Adventures of Ichabod and Mr. Toad: Headless Horseman
- Cinderella: Lady Tremaine, Anastasia, Drizella and Lucifer
- Alice in Wonderland: The Queen of Hearts and Card Soldiers
- Peter Pan: Captain Hook, Pirates Crew and Tick-Tock the Crocodile
- Lady and the Tramp: Aunt Sarah (unseen) and Si and Am
- Sleeping Beauty: Maleficent and Goons
- 101 Dalmatians: Cruella De Vil and Horace and Jasper Badun
- The Sword in the Stone: Madam Mim
- The Jungle Book: Shere Khan (unseen) and Kaa
- Robin Hood: Prince John
- The Rescuers: Madame Medusa and Mr. Snoops
- The Great Mouse Detective: Professor Ratigan and Fidget
- The Little Mermaid: Ursula The Sea Witch
- Beauty and the Beast: Gaston and LeFou
- Aladdin: Jafar (Leader) and Iago
- The Lion King: Shenzi, Banzai and Ed
- The Hunchback of Notre Dame: Judge Claude Frollo
- Hercules: Hades, Pain and Panic and The Fates
- Haunted Mansion: The Hitchhiking Ghosts
Like the show, the film shows several Disney cartoons. In fact, these shorts make up more of the movie than the actual plot with the villains. However, all their title cards are edited out. Among them, three of the shorts are classic ones:
- Mickey's Mechanical House
- How to Haunt a House
- Dance of the Goofys
- Donald's Halloween Scare
- Hansel and Gretel
- When Mickey was about to battle Jafar in his wizard attire there is a direct reference to the dinosaur section of "Rite of Spring" from Fantasia. When Mickey appears on stage and all of the villains gradually turn their heads towards him is very similar to when the Tyrannosaurus Rex appears and all of the other dinosaurs face him. Jafar's defeat at the end is similar to that of Aladdin, where he gets sucked into the lamp.
- Despite appearing on the DVD cover's backside and in the short "Mickey's Mechanical House", Pluto does not make an appearance in the film itself.
- Some scenes from the beginning of the film were later used in the episode "House Ghosts".
- Strangely, some of the characters involved in the takeover of the House aren't actually villains at all, most notably Tick-Tock (a neutral character who spent all of his screentime trying to eat Captain Hook) and the Haunted Mansion ghosts (who couldn't honestly be considered villains by any stretch, apart from Constance Hatchaway, who wasn't yet a character).
- When Goofy calls Donald a "Big Red Bunny", this could be a reference to the "Noid" from the 1980s Domino's Pizza commercials.