Hugo Strange and Catwoman, in order to give the above-seen page image context. If you get seen during Catwoman’s Stealth-Based Mission, Hugo Strange appears in the game over screen. One of them is him holding a scalpel with a Slasher Smile on his face and smugly saying: "I will enjoy dissecting your brain, Miss Kyle!", in his Christopher Lee-esque voice. The way he said it makes it sound like he was eager to slice Catwoman's head open while she was still alive.
The premise itself is based on the idea of a city (or a large portion thereof), being turned into what is, for all intents and purposes, a free range concentration/extermination camp.
Protocol 10. On Hugo Strange's orders, TYGER just starts shooting the place up. Extra horrifying because a fair portion of the prisoners are either in there on incredibly flimsy charges or because they knew too much about Strange's plans. They murder roughly a quarter of the prisoners before Batman shuts them down.
Also, Strange was planning to do this over in Metropolis and Keystone City once he was done eradicating everyone in Gotham. While the plan might not have flown quite as well as it did in Gotham, the thought of having the likes of Metallo, Captain Cold, Parasite, Zoom, Brainiac, and the like locked up in an isolated part of each city then blasted to smithereens, which can be Fridge Horror for those who have read the Superman/Batman arc "Absolute Power". In that storyline, there were many alternate universes, including one where Batman did not exist (due to Bruce's parents living) and in that one, Ra's Al Ghul killed the entire Justice League.
Azrael. He follows Batman everywhere, and if you throw Batarangs at him, he will simply catch them (there's an achievement for doing that, though). Yes: someone who can stalkthe Goddamn Batman. Curiously swinging to the top of a Ferris wheel results in finding some guy in a hood calmly staring you down.
When fighting Solomon Grundy, he's constantly shedding supernaturally huge maggots that attack you. Maggots the size of Batman's foot.
Mad Hatter's mission is twenty shades of disturbing, especially because it shouldn't be. The hats, the tea party, the Alice references, putting a bunny mask on Batman... it should be ridiculous. Except it isn't.
Said bunny mask is shown as a Jump Scare when you struggle to take it off. It's not helped by the eerie similarities between the bunny mask and the one seen in Donnie Darko.
His interview tapes count as well: Hugo Strange giving him free rein to Mind Rape several test subjects who "Nobody will miss" is a pretty horrifying idea. Even worse for the new assistant Strange hired to become his new "Alice".
Those interview tapes, plus the 'Arkham City Stories', reveal more: remember those gibbering lunatics who liked to jump on Batman's back in Asylum? Those are the guys Strange was already testing mind-control devices on.
Harley Quinn has a pregnancy test that says 'positive' in Joker's hideout. Be scared for the third game!
Turns out the test was a false positive. In Harley Quinn's Revenge, you can find a lot more used tests, all of them negative. They have been circled around a crib... with a Joker'd up Scarface. It's safe to say that Harley has indeed, "lost it, big time".
Joker's literal swan song during the credits: "Only yooooouuuuu...", though it's sort of funny in a black comedy sort of way. Which was probably the point.
The Penguin, who finally gets to show off some of the "depraved mob boss" cruelty he's had in the comics. He becomes a Big Bad to rival Joker, and in generally unsettling with his unpleasant mannerisms and the way he brutally tortures the cops he holds hostage.
This gem: "I hope you're breathing through fractured ribs and punctured lungs! And if you're not, then you'd better summon up all your energy and run, because after I finish with the Bat, you're all next!". Sure, slight paraphrasing, but take a chill pill, Penguin.
The Penguin isn't a good boss to work for either, as a few of his henchmen find out.
Penguin: [blows up a bridge] See, I told you it would work. Blow up the bridges and cut off the clown's forces. Easy."
Thug: But, Mr. Cobblepot, we're stuck too!
Thug: We can't get back!
Penguin: And your point is?
Thug: You've left us here with Joker's crew!
Penguin: Try an' take some of 'em down before ya die, son! [disconnects]
Not to mention that his customary monocle in this version is replaced with the butt of a glass bottle shoved into his face in a bar-fight, which can't be removed for the damage it would do!
Regardless of how horrifying Two-Face was in The Dark Knight, Arkhamverse's Two-Face is somehow even worse.
Way worse. Nolan's Two-Face's burned face is nothing compared to the exposed raw flesh of this incarnation.
The Joker's always looked creepier than usual in the Arkhamverse, but this time around his appearance borders on Body Horror: his disease has taken an obvious and terrible toll. After encountering him at the steel mill, the loading screen of his white face and expression make him look like a decaying skull.
Solomon Grundy's partially-rotted face is Body Horror as well.
During the fight with Mr. Freeze, there's a part where Batman is punching him and Victor is suddenly replaced by the Joker, who reaches out and grabs his face during a creepy hallucination.
The glowing bloodlines in Bane's Venom-fueled body, in yet another instance of Body Horror.
For those too afraid to tackle the YouTube link, there is a hidden section inside a boat, that functions very similar to the visitors' center in Arkham Asylum; it's the only part in the game where you are forced into first person mode. The place is very dim and low lit with flickering lights, bugs are crawling all over the place, in front of you is a passed out inmate who is, more than likely, completely screwed up by nightmare gas, and when you approach he will at one point twitch and scream before promptly passing out again, next to a letter to Dr. Jonathan Crane.
While he doesn't make an actual appearance beyond a quick cameo, Killer Croc's name is mentioned by the prisoners throughout Arkham City, some even saying that they've seen him in the sewers, thus providing Paranoia Fuel that eventually culminates in that Jump Scare of a cameo of his, in the sewers. He can smell Batman dying and decides to wait until the poison kills him, then eat the corpse.
If you fail to save the Riddler's second hostage, you get to watch him being roasted alive, onscreen. You can still hear his screams as the screen fades to black.
Hearing the screams of political prisoners as they're being picked on and beaten up on by other residents of Arkham City can be unsettling the first couple of times you hear them.
Riddler's final challenge room, where you must save Anne Bishop. As you progress through the room, you will hear Riddler shocking her at regular intervals while she screams in agony. And every time, once the electricity stops, she gasps in pain and begs you to save her. And all this while he's taunting you about how stupid you are for not solving it faster.
Reading some of the Arkham City Stories unlocked by completing Riddler Challenges (specifically the actual riddles players must scan) can be creepy: one in particular concerned the obscure villain Ratcatcher. When Arkham City opened, he decided to use his rats to smuggle in whatever goods Penguin didn't (mainly necessities like hygiene products, clothing, maybe food, to grant the Penguin some kind of monopoly). Eventually, Penguin decided he wanted to meet his "rival" in the "selling stuff" business: Ratcatcher was dragged, kicking and screaming, into the Museum that Penguin operates out of and was never heard from again.
When fighting Mr Freeze, you can briefly scan him in Detective Mode before his jammer blocks your vision. The creepy part is that while most human characters show up as x-ray skeletons in Detective Mode, Freeze is only a skeleton from the neck up. Just a skull and stump of spinal cord in a robotic suit. This only means that other than the head, the suit can't be penetrated by Detective Mode's x-ray vision. Another detail some people might find unnerving that unlike other enemies, his heart rate drops to single digits while you're fighting him, but climbs back to normal when outside the fight.
The whole ending of the main game can be seen this way. "Highlights" include Hugo Strange being stabbed through the chest in a bloody fashion, Ra's being impaled, Talia getting shot and you face off with Clayface before watching as the Joker dies in a messed up version of Go Out with a Smile.
Combined with Tear Jerker: it's not just the Joker's death, but his final moments leading up to it. Right after his last laugh and his Incurable Cough of Death, his body suddenly starts jerking as he clutches his chest and makes very loud, painful gasps, indicating that the Titan poisoning has completely taken over his bloodstream. The gasps start slowing down and getting quieter as seconds pass, until, at his final gasp, he quietly lets out his long, last breath as he goes out with a smile. Nothing says "painful sadness" like his last moments.
During the final battle with Clayface, one of his attacks involves morphing into a ball with a huge, terrifying face and a gaping black mouth, rolling right towards the player.
His story about his father takes the cake. It starts out actually kind of nice, although the player is likely wary about what's coming next because this is probably not the first time they've heard one of his stories. If it is, players are in for a Wham Line at the end of the monologue.
Calendar Man: "I wasn't real close to my dad, and after my first internment at Arkham we never spoke at all. Seems he wrote me off as a wacko, a loser. So after I was released, I wanted to clear the air between us. The next Father's Day I dropped by his place and suggested we go fishing. You ever go fishing with your pop? Well, it's some fun, let me tell you. The two of us, out on the water, pulling in one whopper after another. Of course, I was doing the actual pulling. Dad was baiting the hooks. You know, with a finger, a foot, an eye... whatever I had left of him. Even today, whenever I eat a nice piece of fish, I feel closer to my dear old dad."
Exploring Gotham City tunnels for a long period of time searching for Ra's al Ghul and the League of Assassins can be creepy as well, when, after finding him and head back to the surface near the subway: you are greeted with a load of bodies piled on top of each other, Holocaust-style. Each of these men were Penguin's soldiers and after Batman took down Penguin and started searching for Ra's, a gang, presumably the Joker's, offed the leaderless clan and executed them all. It's a disturbing sight to behold.
In Wonder City, when you enter the second room upon nearing Ra's al Ghul's lair, you're presented with a roomful of hanged inmates, and a still living one with a sword in his back saying "They're... everywhere!" before expiring.
Even worse, Ra's al Ghul eventually left its citizens to go insane due to radiation from the Lazarus Pit based technology as their civilization collapsed. Now, if radiation from a Lazarus Pit can make people go insane and there is one located right under Gotham and has been there for centuries, then there is now a possible reason for why Gotham is an insane Crapsack World.
Shortly after exiting the gladiator arena in the Penguin's museum, you walk through a hall with fish tanks on the walls. You can't see much in them besides the fish, but turn on Detective Mode and you're treated to the sight of corpses.
In the area before the fight with Mr. Hammer, if you wait around in the area where you can see the majority of the crowd, and not much of Mr. Hammer, one of the inmates will look back to stare at you. While he doesn't do anything, the longer you wait around, the more he'll look back.
During the second visit to Joker's hideout, two Joker thugs can be seen hanging a Two-Face mook over acid, demanding information on his boss. Granted, it's just a horrible situation, but waiting around long enough will prompt one of the Joker goons to recall the last guy they did it to - complete with disgusting explanations on how his skin started to burn before he even touched the acid, including a description of how melting flesh smells. Wait around long enough and you get to see them lowering him into it, only for them to stop and raise him up again (in a "why aren't you stopping this?" kind of way); however, once you do rescue him, he decides toattack Batman.
During (or after) your journey through Zsasz's hideout, if you stop near the floating rafter and turn on Detective mode, you can spot four skeletal figures: one belonging to Zsasz, two are his hostages, and there's a third sitting in the bottom of the flooding and draining room. How it got there in the first place isn't addressed, but then again, knowing Zsasz, it would be almost redundant.
The body is explained in Zsasz's first few calls. He says he has 3 hostages, then Batman pisses him off and he mentions that he would regret that. Nightmarish when you consider that the man who you are playing as, who can kill no one and tries to save everyone, is indirectly responsible for a hostage murdered by a psychopath.
During the first trip to the Steel Mill, we see Harley berating a terrified doctor who failed to cure the Joker, before throwing her to a baying crowd of henchman and calling for Mr. Hammer. The sight of the doctor cowering in terror and the metallic clang of the hammer on the ground as the crowd chants "Hammer! Hammer!" is pretty chilling. Case in point: you aren't going to get there in time. Thankfully, Harley calls Hammer off, but if she hadn't, Stacy would have been brutally murdered with Batman less than ten feet away.
While searching for the stolen Freeze tech, one thug calmly talks about how he once force-fed his own mother to the point of suffocating after she refused to eat it herself. These would have been the guys who went to Blackgate because they weren't insane. Yet they kill family members because of not liking them. The other goon he is talking to is in disbelief about the whole thing, but sounds impressed about what this guy did. Nevermind the cake was already poisoned in the first place.
What makes it subtly more disturbing is the fact that none of the inmates seem to have any problem with this. Real Life prison inmates place matricide as second only to child molestation as the worst crime you can commit. It makes one wonder just how screwed up the Joker's entire gang is.
Harley's Room Full of Crazy in the DLC: a shrine to Joker with incredibly depressing and incredibly disturbing graffiti all over the walls.
If you zoom in on the Joker television-mannequins, they start laughing quietly. It's unnerving to a first-time player. Add that to the background music and the fact Harley sounds more insane than she did the first time and you've got a damned effective opening.
In Catwoman's mission she can choose to walk off with the loot or save Batman. Walk off with the loot and the game ends, with the credits rolling and horrifying radio messages of how everyone in Arkham and Gotham had been killed because Catwoman didn't save Batman. After a while, the scene rewinds back so she can make the right choice.
As Batman pays his respects at the spot where the Waynes had died in Crime Alley, you hear tearjerking music... and the gunshots that killed them, over and over again until you get up. Not only that, there is Strange's message before hand, which details how much fun Strange is going to have revealing Batman's secret and watching him crumble.
Take too long to complete the "disarm the bombs" mission in Harley Quinn's Revenge, and you get to see Gordon watching the building that Batman, Robin and Harley are in explode.
Scarecrow's hidden messages over the radio. Chances are you'll stumble onto them by accident and be immediately freaked out as you try to figure out what the hell that was. But imagine if that actually happened. You're watching TV or listening to the radio in the car and suddenly everything stops... and all you hear is the sound of static and then a monotone voice calmly stating "9 23 9 12 12 18 5 20 21 18 14 2 1 20 13 1 14" and then nothing.
The numbers, when replaced with the corresponding letter, translate into "I Will Return Batman".