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. They speak English, too, saying "Here I come!" before attacking, even in the Japanese audio track of the game.
When Seto leaves the train station through a tunnel, he and PF see the sunset. Nice enough. But off to the right, there are tally marks on a wall. It could be the number of days that another person had been there...or it could be the number of people who've died going the way Seto was about to go. In that case though, who made the marks? This Troper was wary enough by that point in the game to be paranoid about something like that.
After exiting the tunnel, Seto finds the ghost of a little girl in a warehouse. On the walls there are scribbles. Some are happy, like a girl and a mother, flowers, and other things kids might draw...but there are also things like "Mommy" and "I'm hungry" written on the walls.
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.