In one of the earliest levels, you have to dig up a dead little girl to win a game of hide-and-go-seek. The little girl's tree-esque brother innocently carts her still-dressed skeleton around in a wagon for the rest of the level.
She's a really good hider. She's been hiding for a long time.
Even if the random gibbering the the asylum sections doesn't get you, there's also the giant green plant monster "Mother" and all the kids she's transforming, who make you dig up a dead girl to play hide-and-seek, and even the end where it turns out you're just in a coma, but the guy who put you in the coma decides you're not dying fast enough for him and you briefly wake up to see him inject poison into your IV. Couple that with his face then being attached to a black glob of evil and all the horrifying things throughout the game being put together in the final level...
And of course, the part where you're playing as some sort of four-armed cyclops in an insect hive and find the gigantic insect queen who's being fed by having cloned cyclopean children strapped to her immense body and slowly drained of all their blood and nutrients. Sweet Jesus.
Chapter 1. You see a lot of self-destructive, raving loons in a devastated tower (including one who falls off a ledge, screams, and dies as you approach), and an alarm is constantly heard (unless you find it and turn it off), giving the whole scene a sense of immediate, impending danger.
Your character screaming right as the aforementioned falling occurs probably also qualifies.
The loading screen for chapter 7: the morgue level
Then, once chapter 7 actually starts, you're greeted by a dead, frozen woman coming to life long enough to give you some advice on what to do, and then lying back down and going stiff again.
Chapter 7 also has the scariest music in the entire game, so any in-game events, even the ones that aren't inherently scary, have this downright unnerving atmosphere.
Quite a few of the secondary character's facial portraits for when you engage them in conversation. Some of them make make even the non-scary characters (such as Arthur) look unnerving, if not horrifying.
The puzzle where you have to cremate a dead body is probably the most extreme example.
The Nightmare Fuel even starts before the game begins. The installer plays some scary music. Do not install at night!
In the game menu, you can make those creepy eyes look right at you if you move your cursor just right.