Mario sets off the detonator after beating Roy Koopa in the fifth castle, only to see the castle not blow up. He goes to take a look at it... and then the castle blows up right in his face.
His expression is priceless.
In the All-Stars version, Luigi gets an even better one- He just looks at the player with a deadpan expression.
In a similar vein, after beating Ludwig von Koopa in the fourth castle, Mario sets off the detonator to blow up the castle... only for the castle to launch into the air like a rocket and impact against a nearby hill... which then wears a bandage where the castle hit for the rest of the game.
Castle #6 is destroyed by Mario getting a mop. After a few wipes, it's just gone. The dour uneventfulness with which Mario proceeds is what makes it comical.
Also, Mario affectionately calling Koopa's castle, his "Cony Island Disco Palace".
The Mama Luigi Project, a large collaborative project to reanimate the episode. With over 227 artists and animators, most of the remake consists of Deranged Animation, sight gags, and general craziness. Highlights include:
While sinking in the water, Luigi and Yoshi float past a sign reading "Air-Flavored Ocean", Lampshading their ability to breathe underwater.
The Torpedo Ted that chases Luigi and Yoshi during the underwater sequence, at one point, is holding a knife.
When Luigi says "Was it something I said?", he suddenly starts impersonating Rodney Dangerfield, tie and all.
When in Yoshi's stomach, Mario turns on a light and points at a meatball, with a dead Wiggler floating in Yoshi's stomach acid in the background.
Koopa's Wild Take upon seeing the state of his throne room is even wilder. Hell, ALL of his expressions count.
At the famous line, Luigi pulls Mario towards him by the throat, then throws him to the ground, before visibly gagging when he wheezes.
Back in Dome City, Mario is looking at the ground, Luigi is picking his nose, Princess Toadstool has turned into Rabbid Peach, and Yoshi is really angry for some reason.
"Fire Sale": Kootie Pie asks Mama Fireplant if she's been putting on weight.
At the end, Kootie Pie becomes so cold, she cries ice cubes.
"The Wheel Thing":
Kooky: Fasten your seat belt, it's gonna be a bumpy ride! Koopa: What seat belt?
When one of the Monty Moles is being pulled up by Yoshi, Cheatsy states sarcastically, "Don't just help him! Stand there!"; the Monty Moles, not understanding sarcasm, do just that, leading to Cheatsy berating them to help said Monty Mole.
Earlier in the episode, when Cheatsy is singing his Villain Song, the Mario brothers and Yoshi get rid of all the Monty Moles when he's not looking, and they end up being the ones singing the final chorus. It's only after the song's over that Cheatsy sees what happened, leading to a Wild Take. Mario's nonchalant smile and wave when Cheatsy sees them is what really sells it.