* AccidentalInnuendo: Level 2 of Mario Zone is set approximately in the crotch area of a giant mechanical Mario. It's full of... balls. Big, bright balls, spiked balls, bears riding balls. Balls.
** In the first level of Space Zone, there is mound of rocks that resembles [[FlippingTheBird a certain rude hand gesture...]]
* HilariousInHindsight: Space Zone. See VideoGame/SuperMarioGalaxy.
* MemeticMutation: "[[http://www.youtube.com/watch?v=DhB8XG9_5Rc Obey Wario, DESTROY MARIO]]!!!"
* ThatOneBoss: The three pigs in the Mario Zone could be this, seeing as how they each take three hits to defeat, and [[CaptainObvious there's three of them.]]
** Ditto for the boss of Space Zone (who appears to be Tatanga from the previous game), who is arguably right behind Wario as the toughest boss.
* ThatOneLevel: Star Maze. Those stars are going to kill you many, many times.
** While it's expected that a final level would be difficult, Wario/Mario's Castle can be absolutely brutal. While the rest of the game (barring Space Zone) is relatively easy, the Castle is a difficulty spike that can be very jarring for first time players. Combine pools of acid/lava, swinging spiked balls, spiked traps, and several jumps that require almost-pixel-perfect placement, and you've got a tough level. On the plus side, the Bunny Ears power-up makes the stage much easier to manage, provided the player is able to keep hold of it.