Luigi's repeated failed attempts at trying to run away from Sunnycide.
The "L33T HAMM3R BROZ."
The moment Toadsworth sees his past self with Baby Peach, he instantly assumes that his younger self has de-aged Peach into a baby and kidnapped her, and starts chasing him around. Even when he figures out the truth, he just can't get along with his past self. On a related note, their attempts to cheer Baby Peach up are also pretty funny. The Mario Bros even use these to learn new moves.
To add to the hilarity, Elder (present) Toadsworth get more and more aggravated at the physical toll that demonstrating the moves is having on him until he outright sits out of the last tutorial. He spends the rest of the game until the ending in baby Peach's stroller, apparently still in shock of flattening her before said tutorial.
As Baby Bowser is going on a tangent about why the Mario Bros. are technically the bad guys, the baby bros. are busy eating from the massive pile of cookies that baby Bowser had for himself. Then he notices them as they are finishing up the last few crumbs.
After falling into the secret caves under the Koopaseum, the babies attempt to wake up their unconscious adult selves. Baby Mario's solution (as usual) is to hit Luigi with his hammer. This does wake up Luigi, but he (understandably) gets mad at Baby Mario, which causes him to start crying. This causes Baby Luigi to start crying as well and Luigi starts panicking. Mario wakes up to the sound and he gets mad that Luigi made the babies cry. Which causes Luigi to start crying as well. Mario reacts to this scene with exasperation.
The Star Gate's insinuating that Luigi is not pure of heart and cannot go through him, dangling a certain "secret" of Luigi's that the Gate claims is scandalous. Also, it claims that Mario should really eat more healthily than he does.
Some elaboration: The Star Gate declares that Mario's heart is pure and full of virtue and courage, then he goes on to criticize Mario's diet and waistline. It comes across as though the only negative thing you can possibly say about Mario is that he's kinda fat.
If saving upon entering Gritzy Desert for the first time, the save file description reads as follows:
When talking to the toad wandering around in the shop area during early game, you'll find out that reception for their most popular item, the mushroom, ranges from "Rich flavor!" to "Great Texture" to... "Gross".
In the Thwomp Volcano, we finally get to see what a "female" Thwomp looks like... A round boulder with a face bearing crooked teeth and a massive unibrow. That you fight as a boss. Because she and her husband didn't go on what was their equivalent of a vacationnote Jumping into an active volcano.
And then there's her husband, a Thwomp that the bros. climb on to get into the volcano, telling her that she's too heavy for him to carry into the volcano. She then proceeds to violently knock him into the volcano while calling him a jerk.
Just before that, there was when everyone tries to climb the Thwomp in the first place. Luigi jumps up in spectacular fashion and starts dancing. Then Mario helps his past self up and gets on himself. The Marios then stare at Luigi, who stops dancing for a bit before he realizes that he forgot to help his past self get up, and is now trying to climb the Thwomp's face.
Right before entering Shroob Castle, Stuffwell gives everyone a pep talk, which culminates into everyone posing dramatically. Except for Luigi, whose response is a classic Luigi-esque "No". After an intense staredown with everyone else, Luigi reluctantly agrees to go with them.