A typical episode of Toonsylvania starts with a cartoon featuring the adventures of Dr. Vic Frankenstein (voiced by David Warner), his assistant Igor (voiced by Wayne Knight) who always sets out to prove that he's the real genius instead of Dr. Vic, and their dim-witted Frankenstein Monster known as Phil (voiced by Brad Garrett). Before the second cartoon, there is an animated vignette where Igor is on the couch with Phil and tries to fix the TV remote, but in every episode there's a new problem with it (a running gag akin to the couch gags seen on The Simpsons).

After that, there is a cartoon series called "Night of the Living Fred", about a family of zombies. This segment was created by Pulitzer Prize-winning cartoonist Mike Peters. Sometimes, a parody of a B-list horror movie would air instead of a "Night of the Living Fred" cartoon.

After that is a short segment called "Igor's Science Minute", where Igor gives a science lesson (be it a musical piece or a spoken piece) that always ends in disaster.

The final segment is "Melissa Screetch's Morbid Morals", where Phil the Frankenstein monster does something bad and Igor punishes him by reading a tale involving a bratty girl named Melissa Screetch (voiced by Nancy Cartwright of Simpsons fame) who doesn't heed the warnings of adults (usually given by her mother) and suffers the consequences one way or the other for it.