Isaac can wear his mother's brassiere or her menstrual pad as items. Doing so causes the enemies to freeze or back away, respectively. Presumably, they're Squicked out by the sight of it!
Segmented enemies like Larry Jr. will even fall apart when they try to flee. They are so disgusted that it breaks the laws of physics.
When you meet Greed in a hidden room, you can immediately leave through the hole you made without having to defeat him. Hi, Greed. Bye, Greed. If you have X-ray glasses, you can even use his room as a shortcut, running past him without ever engaging him.
The beggar that runs the shell game in the arcade is a jerk. Giving you a giant grin as he takes all your money without giving you the items you want? Seeing the slot machine throw a troll bomb at him is just too hilarious a karma to be true.
The glitches in this game can sometimes cause hilarious reactions from players. Especially since, given the tone of the game and previous games by Edmund Mcmillen, it's difficult to tell if some of them are actually glitches.
One of the random effects of pills is "I Found Pills" with the description "...and ate them." This pill also causes Isaac to get a silly, derped look on his face and make a faint "duuur" noise when you take it.
Another effect of the pills is the "Puberty" pill, which causes Isaac to grow a few scraggly hairs on the top of his head. Then you realize, those are pubes growing on his head...
Any of the secret messages from the Fortune Telling machine. Some of these include cryptic nonsense like "LOOK TO LA LUNA" or "DON'T LEAVE THE HOUSE TODAY". Then there are some that seem to be talking to the player, such as "GO OUTSIDE" or "YOU ARE THROWING YOUR LIFE AWAY". The way the game is just totally fucking with you in this moment is priceless. Oh, the best of all these? YOU ARE WORSHIPPING A SUN GOD.
Some of the boss enemies make some pretty funny expressions, such as Chub's poker face during her intro, War's Oh Crap face when he runs out of stamina, Pin's stereotypical frown (it looks exactly like :c), or even Loki's expressions of pure glee while he fires his 4 way shots.
The fact that of all the Seven Deadly Sins, the only one without a unique sprite is Sloth. Or, in more modern terms, laziness.
One of the between-level dreams features Isaac using the toilet...and discovering there's no toilet paper left. His reaction is the typical "No!" reaction used in other dreams, but it seems more like he's going "Curses!" this time around. Don't worry, Isaac, other people can relate.
Fighting Wrath...while you have the Remote Detonator on hand.