During "Your Fault", there's the hilariously blase way she does her tongue-twisting line about how "the place got cursed in the first place".
Also in the filmed version, the delivery of these lines:
Witch: A giant has a brain. A giant is like us, only bigger...much muuuuuuch bigger...SO BIG...that to them we are only tiny insignificant bugs. (She then proceeds to squash a bug and eat it. She leaves the Baker's house.)
Baker's Wife: We're moving!
Little Red's hysterical, over the top screaming when the Baker takes her cape from her, topped by:
Baker: I just wanted to be sure you really loved this cape!
Little Red: (stomps on the Baker's foot and/or depending on the production, kicks him in the nuts)
Jack's Mother: You need to be careful with your children.
Baker's Wife:(sadly) I have no children.
Jack's Mother: ...That's okay, too...
The princes bickering in "Agony, Part 2", eventually getting to the "thing about dwarves". And on the topic of "Agony, Part 2": "Ah well, back to my wife!"
"On The Steps of the Palace". The sheer wordplay of the whole thing.
In the filmed version, there's just something constantly hilarious about Red Riding Hood's theft of as much of the Baker's wares as possible while the Baker and his wife futilely attempt to stop her.
The Baker believes that his father died... in a baking accident.
Which turns into a pretty great Brick Joke when Red asks about the destruction caused by the Giantess and the Baker immediately answers that it was a baking accident.
Even better when you consider that the character saying this (Little Red) had an entire conversation with a wolf in the first act.
Red Riding Hood's grandmother is usually over the top and hilarious. In addition to the sheer silliness of Red and Granny climbing out of the wolf's stomach after the Baker cuts it open, Granny turns out to be surprisingly sadistic.
Grandmother: *begins choking the wolf, who is already mutilated and writhing in pain* Kill the devil! Take that knife and cut his evil head of! Let's see the demon sliced into a thousand bits! No! Better yet, let the animal die a painful, agonizing, hidious death!
Red Riding Hood: Granny!
Grandmother: Eeeh, quiet, child! This evil needs to be destroyed! Now, you fetch me some great stones. We'll fill his belly with them, and then we'll watch him try to run away! *giggles gleefully*
Baker: *walks away, disgusted* I will just leave you to your task.
Grandmother: Wait! Don't you want the skins?
Baker: Oh, no, please keep them!
Grandmother: What kind of a hunter are you?!
Baker: I'm a baker!
Grandmother: *drags the baker back into the cottage to skin the wolf*
This little bit when Cinderella's Prince has an affair with the Baker's Wife:
Baker's Wife: This is ridiculous! What am I doing here? I'm in the wrong story!
It's entirely possible that Sondheim wrote the musical just so he could include the line "If the end is right, it justifies the beans!"
In the two scenes where characters step forward and state dramatic, foreshadow-heavy advice, Rapunzel's contribution... is to sing. Both times. Right in the middle of the number.
This bit when they're trying to get Jack to give up his cow: