This is based on opinion. Please don't list it on a work's trope example list.
Nightmare Fuel / Castlevania 64
The Gardener, a giant, unstoppable Frankenstein's monster armed with a chainsaw that chases you around a hedge maze that you do not know how to get out of. Attacking him enough times stops him for a second before he gets back up and starts chasing you again. And he has no introduction, he just suddenly runs around the corner and attacks you, totally catching you off guard.
The rose garden. Player character walks into the room and the vampire Rosa is watering roses. She comments how she just waters the white roses. Player character, with a confused look on his/her face realizes just as the player does that the roses are obviously red. Then the camera zooms into the water pail and you see that Rosa is watering the flowers with blood, and that it has stained them red.
The face Actrise makes after being defeated is a bit unsettling.
Despite the dated graphics, the vampires in this game are legitimately terrifying. Especially one creepy boss fight against a vampire that is immediately followed by a fight against the vampire's recent victim, now a vampire herself.
The music in this game manages to build up creepy atmosphere at several points very well.
The reveal that Malus is Dracula. In the bad endings he gets away with none the wiser, and his promise of marriage to Carrie becomes quite sinister once he says it's a binding contract.
The Behemoth is a giant undead two-story tall bull. However, the most disturbing part about fighting it is that when you do enough damage, its hide and flesh melt off, leaving behind still moving bones, and pulsating internal organs that somehow still remain in there as it keeps coming at you.