There are also the countless sentient carrots being held captive in the refrigerator, for certain routes. If you talk to the refrigerator, he'll sing about how those carrots are all going to be used in a salad.
The kitchen appliances all seem to be singing in a minor key, which gives the whole experience an unsettling feel.
The trees in the first game, especially in the scene where they steal all of your stuff.
Darkness. Although Dark Is Not Evil, just lonely, he still managed to traumatize many children.
The plank of wood sinking after Sam and Otto look away from it. It made quite a few kids think that Otto would eventually sink too.
You Are What You Eat From Your Head To Your Feet
The fact that the S.S.A.M. (Snacks and Sweets Aggressive Majority) at the beginning of the game gives a false sense of security that they seemed friendly at the party only to suddenly turn on Pajama Sam, yelling to throw him to jail just for mentioning that he didn't want to spoil his dinner.