- Moose Hunters (1937; debut)
- Window Cleaners (1940; known as Barrington the Bee)
- Home Defense (1943)
- Inferior Decorator (1948)
- Bubble Bee (1949)
- Honey Harvester (1949)
- Slide, Donald, Slide (1949)
- Bee at the Beach (1950)
- Bee on Guard (1951)
- Let's Stick Together (1952)
- Beezy Bear (1955; cameos on title card and inside a jar)
- The Mad Hermit of Chimney Butte (1960)
- The Ranger's Guide to Nature (1966; cameo)
In Mickey Mouse, Spike returns in "Bee Inspired" where he repeatedly frightens Mickey, keeping Minnie from finishing her painting. In the end, they become friends and Spike saves Mickey from an angry swarm of his companions by giving a dramatic speech (all in buzz language with subtitles). He would also appear in "New Shoes" to torment Goofy while stuck in Donald's body with the rest of Donald's recurring tormentors.
Spike the Bee made a handful of appearances in Disney comics. In the 1948 Inferior Decorator he attacks Donald's house with a whole swarm of bees because Donald's new wallpaper represents flowers.
Spike would be used for Epcot's 2018 Flower and Garden Festival as the host for the "Spike's Pollen Nation Exploration" scavenger hunt game. Twelve figures of Spike were placed around gardens throughout World Showcase to mark the different spots.
- The Bee enemy design in DuckTales Remastered resembles Spike's original design from "Window Cleaners."
- He has stung multiple times meaning he's a bumblebee as honey bees can only sting once before dying.