"Pilot". Pure Adult Fear in Mary's voice when she realizes it's not John in her baby's nursery. "SAMMY!"
Jessica's blank, bloody stare when she burns on the ceiling.
"Wendigo". It's a creature that attacks in the dead of night in the middle of an isolated part of the woods. Oh, and it can unlock doors and get inside your house, and in the episode, you never see it from the front standing in the light. You only see the the characters' reactions to it when it appears. Good luck and sweet dreams if you live in Wendigo territory.
"Bloody Mary". Don't turn out the light, don't go to the bathroom. Don't say anything in the bathroom, because you might accidentally say 'Bloody M'—eep.
Shifter!Dean tearing his skin off in "Skin". The worst part may well be when he pulls out his own teeth. "Hey Man Nice Shot" just makes it all the more effective.
Also, Shifter!Dean chasing Becky through the house and eventually tying her up, with the implication that he was going to rape her.
"Bugs." Bugs in the shower. Bugs all around you if you fall into a pit by accident. Bugs crawling in your ears, in your eyes, swarms of bugs...
"Home" and the dreaded cymbal monkey clanging as the poor repairman's arm gets caught in the garbage disposal unit.
"Asylum" features a few seconds of a random ex-patient twitching writhing violently in a straitjacket. Sam and Dean finish talking about something and walk away, camera pans out, and then it's like something straight out of Silent Hill.
Perhaps the most disturbing part is what he says before it happens:
Stepmother: Max, I'm sorry!
Max: No you're not. You just don't want to die.
There's a lot of creepy Eye Scream in this show. In "The Benders," Dean gets tied down and interrogated by the murderous family, who threaten to burn his eye out with a hot poker. Then the father told little pint-sized Missy Bender to keep watch on Dean—which she did by holding the point of a knife about half an inch from his eyeball.
It might be Fanservice for some, Fan Disservice for others, but the scene in "Devil's Trap" where Dean is bleeding to death (HIS ORGANS ARE MAKING SQUELCHING NOISES) was made even worse by the "Daddy, please...", with him crying and the blood running freely from his mouth and the demon letting John come back up to make him watch...
When he reveals himself in his human identity, he removes his sunglasses to reveal creepy white eyes and his smileliterally stretches ear-to-ear right before he goes invisible and starts attacking. It's a really good thing he isn't wearing his clown outfit at the time or it'd be so much worse.
Dean being roofied into putting the gun to his head in "Simon Said". Finding out about his suicide/death-seeking nature just makes it a hundred times worse.
Webber: I want you to listen to me very carefully, okay? When we're done here, I want you to head over to the edge of that dam, okay?
Webber: And when you get there, you're gonna think you can fly. And you're just gonna step right off. (strokes her face) You can fly, can't you?
Tracey: (starts sobbing again) I-I think so.
The ghost with the slit throat in "The Usual Suspects" trying to talk.
Knowing what one goes through near the end of their time as seen in "Crossroad Blues" (and later personally by Dean in "No Rest for the Wicked"). If it wasn't bad enough that you'll always be seeing the faces of everyone you see suddenly take on the appearance of rotting corpses as a reminder that your time is almost up, you also have to eventually face those damn hellhounds...
It has to be said, this show has a disturbing ability to make near-rape scenes a thousand times scarier, like with the man about to rape the woman ("I'm sorry! I'm sorry, it's just I've never done this before.") in "Houses of the Holy".
The near-rape scene in "Born Under A Bad Sign". Maybe it's because we don't know Sam is possessed at this point, maybe it's because it's one of the leads doing the near-raping, maybe it's the size difference, maybe it's because Jared Padalecki is scarily good at being menacing and Alona Tal was fantastic at acting terrified... There's a subtle but very noticeable contrast to how he normally acts - the way he hunches just a little and is generally the non-threatening of the duo, and all of a sudden his body language is a bit more aggressive and his smile just a little too gleeful, almost a Slasher Smile compared to his usual quick, wide, innocent one. The scare comes at least in part from the fact that you are aware that the 6"4" Sam could easily take down the petite, untrained Jo with no real effort just before he proceeds to do so.
"What Is And What Should Never Be": djinns have been tricking people into staying in their own heads, happy for "years", whilst outside they're slowly dying. On the other hand, imagine if Dean's dream was real; what if Sam had had to watch his brother kill himself, raving on about monsters and how the whole world is a dream?
The part in "The Magnificent Seven" where a hunter was brainwashed to down a jug of drain cleaner while the titular seven demons roared with laughter and his wife hysterically pleaded him to stop was possibly the most disturbing scene in the whole series
It's the "poetic justice" bit that makes it even more disturbing, because you can't help but understand why the demons would love it so much—it's rather like how Bobby got posessed!Sam to drink beer with holy water in it.
"The Kids Are Alright"—not the Monster of the Week, but the image of a mother, with complete, cold calmness, driving to a lake with her daughter, getting out of the car, locking the doors, and then releasing the emergency brake. Finding out what the kid really was made it less scary.
Sam, after Dean's temporary-real death in "Mystery Spot". Completely emotionless, driven, almost robotic. Impressively precise in a sociopathic way (it's mentioned he took out a ton of demons and a vampire nest in the intervening time), but it's still disturbing the way he patches himself up without a sign of emotion.
What about almost the entirety of "Time Is On My Side"? You've got a guy's guts literally spilling out, another guy being shown his still-beating heart and Sam's puppy dog eyes nearly being gouged out with an ice cream scooper!
The final moments when Bela realizes just how screwed she is. The smug overconfidence has completely disappeared and she becomes like a scared little girl. It gets even worse when the clock reaches midnight and we hear the horrible howls of the hellhounds and all Bela can do is look out the window, knowing that she cannot escape.
The very end of "No Rest for the Wicked": Dean chained up in Hell with hooks in his side and shoulder.
Worser and worser, Dean being torn apart by hellhounds.
Little Girl Lilith. Despite the complete horror of a cute kid killing her pet and her grandfather juxtaposed with her wanting to be pushed on the swings or having birthday cake every day, what about when the little girl's NOT possessed anymore? Not enough therapy IN THE WORLD to get over what she went through.
The razor blades in the candy in "It's the Great Pumpkin, Sam Winchester".
A few minutes later in the same episode, the potential HalloweenShout-Out with Jenny getting her face boiled in the now-scalding apple tub.
Anna crying "Her face!" while looking at Ruby in "I Know What You Did Last Summer" sends the imagination into overdrive. The results can be nightmare-inducing.
"Family Remains". The brothers believe a family who just moved into a new house is being haunted by a ghost. Turns out the "ghost" is a human girl who lives in the walls. Most episodes, you can calm yourself down by reminding yourself that monsters aren't real, but not this time...
In that same episode, when it's revealed what happened to Buster the dog. Heck, the savage smile on their faces as they tore a live rat apart with their teeth.
"After School Special": The boy jamming the Jock's fist down a food processor.
The sex scene with the siren in "Sex and Violence": it's a beautiful naked woman until you see its reflection as a lumpy, hairless, gray-skinned thing with a snake-like nose and a rotted mouth. Imagine having sex with that. Imagine being put under its spell and tricked into murdering your loved ones, before it leaves you behind to spend the rest of your life in prison with the realization of what you've done.
Dean torturing Alastair in "On the Head of a Pin."
One of the worst moments came when Dean forced the salt down Alastair's throat. Considering that eating ten or so table spoons of salt could kill you within an hour.
When Alastair gets loose, Dean goes completely limp the instant Alastair gets a hand on him. He's not even trying to resist. Thirty years' worth of conditioning, huh?
The guy microwaving his head to commit suicide in "It's a Terrible Life".
"Jump the Shark": The ghouls slicing Sam up while eating him alive. Gross, and Squick.
Azazel's slaughter of the nuns in the "Lucifer Rising" teaser.
Castiel in "The End": While not outright terrifying, the Broken Angel-ness starts bad (normally straightlaced Cas organising hippie love-fests?) and just gets worse (normally straightlaced Cas turned organiser of hippie love-fests... who is entirely unruffled, telling past-Dean about how he's effectively human and takes amphetamines as "a perfect complement to that absinthe", while on the way to what he knows is his death?). Unsettling, even by Supernatural's standards.
Dean: Are you... stoned?
Cas: Generally, yeah.
There is something deeply unsettling about Lucifer in general, but especially when he's walking about wearing Sam. It hits the Uncanny Valley in so many ways.
Lucifer any time really. First, he isn't the 'devil' most people perceive. He's brutally honest, straight up with the Winchesters (and especially Sam) about his plans. Problem is, there's nothing they can do to stop him. Even the Colt, which was said to be able to destroy just about anything, can't kill him. He's just a really nice guy who just happens to have the end of all human life on his agenda.
Hammer of the Gods: A collection of the most powerful pagan gods are so terrified of Lucifer that they meet together to create a plan to stop him. This includes Odin, Kali, Baldur, and Mercury (the Roman form of Hermes.) What happens when he shows up to the gathering? A one sided slaughter the likes of which is rarely seen in Supernatural.
Death. When first we hear of the Four Horsemen, it's assumed that they're demons. This is very much so not the case when it comes to Death. The guy scares the ever-living crap out of Dean Winchester, who takes crap from NO MAN.
"My Bloody Valentine" and especially that couple at the beginning was nightmare fuel in its pure, distilled form; none of the Horsemen are family-friendly, but Famine is a very special case.
According to an interview even Ben Edlund, the writer, was disgusted.
The scene at the end where the guy is lying dead in the deep-fryer. His whole upper body was in there. The implication being that, since he was earlier shown sticking his hands in the fryer to get to the food within, once he either ate his deep-fried hands or lost use of them, he stuck his face in to eat more.
Watching Sam slowly falling apart over the episode as Famine's influence makes itself known and his addiction to demon blood starts coming back to haunt him can be particularly unsettling. Especially when the two demons show up to capture him and he completely loses control.
"Two and a Half Men": A shifter poses as women's husbands while the husband is out of town, has sex with them, and comes back for the baby nine months later. This has resulted in divorces in some cases. And he is perfectly willing to kill the woman and her husband to get the kid back.
"The Third Man": the effects of the Staff of Moses on the dirty cops—turning into sacks of blood, filled with boils and locusts eating out their brains.
Death putting Sam's soul back in "Appointment in Samarra" seems a little rape-y upon watching it again.
"Unforgiven": Arachne's disfigured face. You can't tell if Arachne come that way or if they were from burns. And don't even get me started on the eyes. And since Arachne apparently breed, what did Roy mean when he told Sam he was considering turning him?
"Mannequin 3: The Reckoning" had possessed Murderous Mannequins stalking people then killing them. The worst part was when they moved on-screen. At least one of the wikis for Supernatural changed its first recap picture for its page on the episode (that of a mannequin smiling after killing someone) because it was a little too unsettling for some people.
"... And Then There Were None": The hissing parasitic "Khan worm" that crawls into your ear and makes you kill people. At least you can see 30 foot monsters; this thing can hide and strike from anywhere. Plus it looks nasty.
Castiel's vessel melting in "Meet the New Boss" and the scene where he unbuttoned his shirt to reveal Purgatory souls are clawing at him from the inside, trying to get out. Becomes even worse if you allow yourself to think of how many of said souls have encountered and been killed by the Winchesters, how many of them see the Winchesters and want revenge...
Season 7 introduces the Leviathans, who spread out into the water supply, infecting anyone who comes in contact with it. Think twice before you take that next drink from the water fountain...
Individual Leviathans have been shown to effortlessly kill angels in a truly horrific way in "Reading is Fundamental".
The ending of "The Girl Next Door". The Leviathan taunts its victim and says "Plain old people taste fine, but everything is better with cheese", then upends a pot of melted cheese onto his head and, while the victim screams, reveals his Leviathan-face right to the camera as he moves in to eat the dying victim. Repulsive.
Bibbing. A Leviathan that has pissed off their boss enough gets a bib put on them and is then forced to eat himself. It has got to be one of, if not the, worst ways to die the series has depicted, even if it can only happen to the bad guys.
From Bad to Worse: Leviathans are nigh-invulnerable and regenerate when injured. Now think about that. You'd be trying to eat yourself faster than you can heal and, if you were too slow, would have to do it again.
The cursed teapot in "Out With the Old" causes a woman to pick up a super-heated metal teapot with her bare hands, then pour the super-heated water into her own mouth. What makes it worse is that you can see her horrified expression as she pours.
The Leviathans' plan. Unlike other evil plans in the show, that tend to focus only on supernatural aspects, Dick Roman is manipulating numerous parts of everyday life. He's taking control of the media, food companies, and more. Why? So he can manipulate everyone on the planet into becoming stupid, complacent, overweight sources of food for the Leviathans and set up slaughterhouses so every human in the world can be used to make a massive meal for the Leviathans. Oh, and he's doing it in ways most people aren't even aware of until it is too late. Once you bite any of the tainted food he's put out there, it is so addictive, it is probably too late for you. But at least you'll become so stupid you probably won't care you're being eaten. To make it worse, as of "There Will Be Blood", Dick is putting chemicals to change people in every food with high fructose corn syrup, so almost every piece of food you can buy will turn you eventually into a Leviathan's meal.
The scariest part of all this is that they consider humans (over 6 billion of us) to be a limited resource!
Sam's hallucinations of Lucifer and the Cage. You'd think the meat hooks, chains, and constantly being told that nothing around you is real would be bad enough. No, the visions have to get so bad that Sam is incapable of sleeping, has to be institutionalized (however briefly), and comes close to dying of sleep deprivation. It also veers into Fridge Horror territory when Lucifer does things like call Sam "bunk buddy". It makes you wonder what exactly happened down there.
Most of Crowley's actions in 'A Little Slice of Kevin'. Torturing Samandriel, blowing up one of the future prophets leaving Kevin covered in their blood, and cutting Kevin's finger off. This episode proves that while Crowley may be Affable, he is most definitely EVIL.
Keep in mind, one of those future prophets covered in Ludicrous Gibs is a little kid.
Also, Castiel's situation regarding Naomi's 'little chats'. Not only is he being forced to spy on the Winchesters against his will (literally, he's compelled to answer Naomi's questions while in her office), but he doesn't even remember doing it.
It gets worse in "Torn and Frayed", when he flashes back to a time when Naomi strapped him to a chair and seemingly lobotomizing him.
Chuck's fate, according to Castiel. He is dead and we don't know why or how. It's horrifying when you think what Crowley did to the OTHER prophets, including Kevin. Did Crowley torture Chuck to death? Did he just kill him? Chuck was portrayed as weak and frightened in most of his appearances. Less horrifying when you watch the interviews with his actor and Castiel's actor, and they both confirm that Chuck was never a prophet in the first place, but actually GOD! One wonders if Crowley even tried to confront Chuck and what happened when/if he did. Hilarity Ensues when King of Hell tries to torture God?
Actually, I think Chuck's "death" was actually him going... somewhere at the end of "Swan Song". He isn't actually dead, he just left. Plus, since Cas didn't know Chuck was God, it's easy to see why he'd think Chuck died...
In the bible, prophets ascend straight to heaven when their work is done, without dying. Some of them become angels (Metatron used to be a human prophet according to most angel lore) but they do just vanish. I don't think anything horrific happened to Chuck, he was just done.
The Monster of the Week in Larp and the Real Girl, the way it just pops out of nowhere provides a genuinely terrifying Jump Scare, and the deer-skull it uses makes the head seem unnaturally long. Of course this gets a little less ruined when it's revealed to be a fairy, in the form of Gilda who Charlie has a teeny bit of a crush on.
The opening of "Goodbye Stranger" in which Naomi has apparently been forcing Castiel to kill copies of Dean over and over again as a conditioning strategy. It's bad enough that the first thing the viewer sees when the episode begins is Castiel dispassionately killing a pleading Dean, but when the camera pans up to reveal a room filled with a huge number of Dean-corpses, the situation becomes even more disturbing.
When Hell is re-revealed in Season 8. It's no longer just a abyss of smoke and chains, its a literal torture dungeon where you're tormented by demons taking the form of people you loved. One woman was so broken by it she kept saying 'You came, I prayed for it, you came, I prayed for it' to Sam. All with the sound of souls being eternally tortured.
Actually, the re-introduction is even worse than that, when you remember that Crowley previously mentioned that after taking over, he changed Hell into an endless corridor where people are forced to queue for all eternity. Now it's looking worse than ever before, cruel and twisted just like Crowley himself has become.
What Naomi and her angels do to the Biggerson's diner that Castiel arrives in. Everyone is massacred except for the waitress, who is sitting on the ground with her back against the counter and HER EYES BURNED OUT AND BLACKENED and whimpering over and over in a pleading voice, "You need to stop. Please. You need to stop."
The moments leading up to it. Castiel is rapidly flitting through various Biggerson's diners around the world, all with the sound of people laughing and having fun. Then the sound just stops. He looks down to a table covered with blood, then turns to see the entire restaurant he was in just minutes before, completely massacred.
Crowley in general is nightmarish in season 8. Previously, he walked a fine line as Affably Evil, but now he no longer has any need to broker fragile alliances built on mutually assured destruction with the boys against Lucifer or the Leviathans: he's the last Big Bad on the playing field as of season 8, and he has no reason to hold back. Need an example? Check out the climax of the season, where Crowley intends to systematically slaughter everyone that Sam and Dean ever saved, in order, to get what he wants. This Evil looks a lot less Affable now.
Hael goes from being a cute, quirky angel who built the Grand Canyon herself to a fucking monster. Seeing her seething, covered in blood and glass, ranting about how she'll "make Lucifer's fate look like child's play" is horrifying.
Abaddon in her very first episode of Season 9 has proved she is Nightmare Fuel. She wants war with humanity, for Demons to rule the earth, and is prepared to string up hunters just to get Dean's phone number, and unlike other Demon leaders like Lilith and Crowley she's willing to get with the times and equip her soldiers with assault rifles and wear kevlar body armour herself. Demons with assault rifles. That alone qualifies as Nightmare Fuel. And even worse is what she wants from Dean, Abaddon wants Dean as her new meatsuit, Squick factor of a girl being inside Dean's body added onto what she plans to do if she gets her hands on him,
Abaddon: Oh. Well... you know... I've loved this body since the moment I first saw it. You're the perfect vessel Dean, you give a girl all sorts of nasty ideas. So go ahead and play hard to get, and I'll peel off this no demons allowed tattoo, and blow smoke up your ass.
Dean: Oooh, well I gotta tell you between you and me, it is a horror show up there.
Abaddon: It can get worse, trust me because once I'm on top I'll make you watch. And I'll use your body, have you ever felt an infant's blood drip down your chin? Or listen to a girl scream as you rip her guts out, because you will. You and me lover, we'll have a grand old time.
Dean slowly slipping into a homicidal mindset after getting the First Blade.
It gets worse. Perhaps the most horrific moment in Supernatural: Demon!Dean is born.
The Comic-Con trailer for Season 10 finally reveals a hint of what Demon!Dean will be like, and he's freaking terrifying. Stalking Sam through the bunker, trying to kill him with a hammer, and all the time taunting him like every Demon enemy the Winchesters have ever faced has done to them. Also scary because this trailer confirms that the old Dean is gone, and he may not be coming back.
From 10.1, Dean fighting the Jerk Ass boyfriend outside the bar. He roughs him up, yeah, okay...but then he keeps going. And going. You can practically hear the man's skull cracking, and Dean just keeps going. By the time he stops the poor bastard is barely conscious.
There's something skin-crawlingly freaky about the combination of a demon with a jolly old Slasher Smile (particularly if they have glowing eyes), just because you know they're contemplating how many fun ways they're going to torture you to death. Yes, Azazel, I'm talkin' to you.
The anime version can get away with a lot more gore than live-action, and the creators knew it. Notably, the adaptation of "Devil's Trap" cut out the car crash at the end since Dean getting tortured by Azazel was sufficient to hospitalize him.