The entire Fifield and Millburn death scene. The tension when the creature first appears is bad enough but when it finally makes its move the scene quickly descends into unfiltered terror. It bites Millburn's hand and proceeds to wrap around his arm so tightly it breaks it. Fifield panics at first but then cuts its head off, only to get acid blood in his face. The creature immediately grows a new head, buries itself into Millburn's arm to get into his helmet, and then forces itself down his throat. All whilst Fifield's helmet melts onto his face.
"CUT IT OFF! CUT IT OFF!!!"
The now-infamous scene of Shaw's self-administered emergency surgery. The tense buildup to the eventual cutting of the stomach and removal of the thing she "births" is bad enough, but it gets even worse when it bursts out of its sac in the Medpod's claw inches from her face and starts trying to attack her midway through the surgery.
David: Well, doctor... it's not exactly a traditional fetus.
For bonus Squick points she even rips the umbilical cord out with her own hands.
Bonus creepiness points for this happening to the main character. No one is safe!
The fact that this scene is so utterly claustrophobic!
Not to mention if you already had a crippling fear of squid and octopus before seeing this film. Getting attacked by an alien squid? Bad. Finding an alien squid buried in your abdomen, and having to cut it out yourself? Worse. Cutting it out, and then being stuck in a tiny plastic coffin with said alien squid while it thrashes around wildly just inches away from your face? GAH!
The bit when the amniotic sac bursts open and the amniotic fluid spills onto Shaw as she lies below the writhing, aggressive and clearly angry creature.
A 15-year-old teenager in Australia passed out and had a seizure during this scene and was rushed to the hospital. This sparked a discussion on the rating of the film in Australia, which had been lowered from an "MA 15+" to a more lenient "M" before its release to allow younger teens to watch the film.
There's also the sudden swelling of her stomach, combined with sound effects.
For anyone who's had a c-section or hysterectomy, the amount of running Shaw does immediately after her self-performed surgery is horrifying. In real life, recovery from both procedures is intensely painful if not managed with rest and medication. While Shaw had painkillers, she had to be Made of Iron to keep going as she did.
The scene where the infected Holloway looks in a mirror... and we get an UP-CLOSE shot of a mini tentacle growing from his eye.
The potential for an apocalypse. Not just on Earth but across the galaxy given the nature of the monsters the Engineers create.
The attempt to revive the Engineer's head.
The Xenomorph's long awaited appearance. It's onscreen for less than a minute but makes it clear that despite everything we went through it's about to get a whole lot worse.
The scene where Weyland, Shaw, David, and a few others wake up the Last Engineer. Said Engineer is not amused.
The beginning of the film with David. He's been on the ship for two years with limited stimuli and no interpersonal interactions beyond dreams. Consider him going through the motions, repeating the same directionless routines, alone, for that long.
Fifield's return as a horrible, swollen-faced, super strong zombie who brutally slaughters several crew members with his bare hands, including one unfortunate soul whose chest he jumps on so hard that it causes the guy to vomit blood everywhere, before smashing his face into pulp with his fists. It doesn't help that they only manage to stop him by setting him aflame, pumping multiple bullets into him, and finally running him over with a truck twice. A terrified crew member even shoots a final jet of flame at his burned corpse post-mortem, just to make sure he's down for the count.
The way he walked back to the Prometheus was also horrifying, reminiscent of TheExorcist.
Some might find the original design for his mutated form even more frightening. It resembled more of a human-xeno hybrid where his skull was extending backwards.
The Proto-Xenomorph "Deacon" is even creepier than most Xenomorphs since it's already human-sized when it emerges. It's also noticeably more humanoid than other Xenomorphs, which just makes it worse.
The Engineer being attacked by the Proto-Face Hugger. That thing grabs him and he's struggling against this giant starfish tentacle monster Eldritch Abomination that forces itself down his mouth.
The Engineers themselves. Imagine finding out that humanity was created by super-advanced aliens, and that these aliens left signs clearly indicating that they wanted you to come and meet them. You head out there, eager to learn from these beings, wanting to study them, their knowledge, and their technology, and to have two species meet in peaceful contact. But when you arrive, and finally meet these beings, you discover that they're not peaceful. They attack you on sight, try to kill you, and clearly mean to destroy earth, the human race, and everyone you love and care about. And the worst part? No matter how much you beg for answers, no matter what you say, they won't even tell you why they changed their minds and want to kill you. You are just insects to them, parasites to be wiped out, and nothing more.
Humanity may very well be this for the Engineers. Imagine that your experiments, which you've been keeping tabs on and plan to eventually dispose of, have taken on a life of their own, figured out their origins and discovered where you are. Not only that, they have the means to get to you. It's as if the parasites you're trying to wipe out could now literally walk up and threaten your very existence.
David's expression, when he tells Shaw he was looking into her dreams while she was in stasis, is one of the creepiest things in the entire movie. And that's saying something.