Toward the end of the game, Mario and company are attempting to rescue Peach from Bowletta. Fortunately, Toadsworth had packed an extra dress for Peach in case of Clothing Damage. Mario's solution? DressLuigias Peach, then attempt to pass him off as the "real" princess. Luigi goes the extra mile; he mimics Peach's voice with surprising accuracy, and even covers his face with his hands to hide his mustache...until he is in Bowletta's cruiser.
Bowletta: By the way, Princess...why are you covering your mouth with your hands?
Fawful: Do you hide something?
Bowletta fakes Luigi out, and Luigi uncovers his face.
Bowletta: Fawful... Did you see that?
Fawful: Yes. Mustache.
Bowletta: ...Fire at will.
Hermie III thinking that Mario is Santa Claus because of his mustache and red clothes.
The whole sequence after Mario's consumed the Invincishroom. Luigi moving very slowly due to fear and being hypnotized into thinking that he's Mario in particular.
Mario running around in his towel.
Practically every time Fawful is on-screen.
Try flubbing the Bros. Attacks. The expressions are hilarious.
Having the brother in back use the hammer or hand attacks before they learn the proper combo for them leads to a hilariously angry reaction from the brother in front.
After Mario gains the ability to be shrunken by Luigi smacking him over the head with a hammer, you can do the above to Mario and then attempt to perform a high jump. Naturally, since Mario is shrunken, this'll fail, but during the brief moment while Luigi is above of Mario's head, you can make him jump with the expected results. Not only is this hilarious in itself to begin with, but you can also keep juggling poor Luigi for as you like AND also get coins out of it for as long as Luigi has more than 1 HP left, which he keeps losing as his brother lovingly pounds the everloving crap out of his crotch hard enough to keep him suspended in midair.