Mighty Jill Off incorporates several BDSM themes. It stars a submissive lesbian named Jill who has a boot fetish. She is kicked to the bottom of her Queen's tower for acting like a "greedy slut". Jill attempts to climb back up the tower and return to her Queen; once there, she is given affection, constricted and gagged, and made to do it again.

Some of Mighty Jill Off's gameplay is borrowed from the 1986 arcade game Mighty Bomb Jack as a tribute. Players control Jill in only three ways: moving, jumping, and slowly descending. In the air, players may cause Jill to slowly descend by rapidly pushing the jump button while pointing in the direction that they want her to go. Players can switch direction while they do this. There are three types of obstacles: spikes, fire, and spiders; the latter can move to attack Jill when she comes close. Coming into contact with these obstacles kills Jill instantly, forcing her to start at the most recent checkpoint—typically located at a change in room. There is only one level in the game, but with several rooms in it. The screen scrolls vertically with Jill as she ascends up the tower. Each room transition is demonstrated by a change in the room's background colour. Jill has infinite lives, allowing players to play as long as they like without having to start the game over