The plot, like that of many similar arcade games, is simple. After stealing a giant diamond from Springfield Jewelers, Waylon Smithers bumps into Homer Simpson, whose family was conveniently walking down the street. The encounter knocks the diamond out of Smithers' hand, and Maggie Simpson snatches it in her mouth (in place of her pacifier), prompting Smithers to grab Maggie and run off. The Simpsons must give chase, but hundreds of goons in matching suits stand in their way.

Bart Simpson has been sent to his room and it’s up to you to help him escape and collect the items he’ll need to rescue his hero Krusty the Clown. Along with the standard side scroller jump/duck, Bart is armed with a variety of weapons such as a slingshot, spray paint, and water balloons which he can use to dispatch the space mutants, basement ghosts, and other uncool things getting in his way.

There are six levels for exploration including a spider infested attic, the Springfield Mall, and a twisted Itchy & Scratchy dream world. Ultimately, when Bart manages to complete all the levels, he is taken to Krustyland for a final showdown with his nemesis Sideshow Bob.

Bart vs. The Space Mutants is a platform game where you play the role of Bart Simpson (from the TV show The Simpsons) and must stop the Space Mutants from invading Springfield.

On each of the five levels, Bart must collect (or get rid of) the ingredients that the Space Mutants are planning to use to build their machine, such as purple objects or balloons. He also has to collect enough "proof" of the aliens existence (brown coins left behind when they are jumped on), so his family members will help him when he meets a boss (characters such as Nelson and Sideshow Bob). This ain't be easy, since the Space Mutants are "using" human bodies as disguise. In order to discover who are the real Space Mutants, Bart must use his X-ray Specs.

The ways Bart can get rid of those objects sought by the Space Mutants are numerous. For instance, in the first stage, Bart must get rid of purple objects. He can dye them red, cover them, wash them, etc. Some even involve a little puzzle solving, like playing a trick on Moe to make him get out of the bar.

Bart has some coins also, that can be used in many ways throughout the game, like buying things, getting devices operating, etc. Those coins are essential to solve some puzzles in the game.

Krusty the Clown's Fun House has been overrun by rats. You must help him to herd the rats into trap machines to clear them out. In order to do so, Krusty must manipulate his environment to set up pathways so that the rats are headed in the right direction. Objects that Krusty move around include blocks, fans, and pipe pieces.

The trap machines are operated by other recognizable Simpson's characters: Bart, Homer, Sideshow Mel, and Corporal Punishment.