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Professor Nefarious is the Sleuth's arch-enemy. Despite being a criminal genius, he believes the Sleuth is a genius detective, never realizing that the Sleuth is totally clueless and that the Sleuth's assistant Mickey Mouse is the one who actually foils him.

Professor Nefarious sees himself as a "teacher of crime" for his three henchmen-pupils Fliplip, Sidney and Armadillo. Their hideout is a rundown townhouse with the words "University of Criminal Sciences" written on its front door. While Nefarious is reasonably smart (although his own megalomania sometimes hinders his plans), his three accomplices are thoroughly inept comical villains. Mickey and the Sleuth imprison the gang at the end of each story, although Nefarious himself generally manages to escape.