This is based on opinion. Please don't list it on a work's trope example list.
Nightmare Fuel / Fragile Dreams: Farewell Ruins of the Moon
Many enemies and places qualify, but the hands coming out of the mirrors in the subway restroom at the beginning of the game seem to be first scare factor for a lot of players.
Other significant ones includes ghosts of children who only have legs and is just a wisp from the waist up, complete with creepy giggling.
Seto enters a trashed room filled with boxes and other post-apocalyptic junk towards the end of the game. For those using one of the regular flashlights, there is nothing remotely disconcerting about this room. However, if you were using the flashlight that allows you to see hidden messages written on the walls, you will suddenly enter a room where literally every surface has "I don't want to die! I don't want to die! I don't want to die!" scribbled over it in an increasingly desperate hand. If you scare easily, do yourself a favor and don't enter the room with that flashlight.