The Head of the Navigator. It's exactly what it sounds like, a mummified head kept alive by magic. Even the more cartoony special edition repaint of its face is still fairly disturbing, and it wears a necklace of eyeballs to boot.
The extremely unsettling catacombs inside the giant monkey head, which are composed of body parts, giant mushrooms, and lava. The thing about these is that they randomly generate and form a maze, and you can't navigate them without the navigator necklace, so if you don't know this you can be going round in circles forever (although it will usually regenerate the room in which you came in after going through a few doors).
The game does a great job mixing creepy creepyness with familiar and funny creepyness. Still, almost everything that happens on the graveyard on Scabb Island happens to be Nightmare Fuel.
The game overall has a very surreal and bizarre feeling to it, especially if you don't know the infamous Twist Ending. Odd things like the concrete maintenance tunnels running under the islands.
Cant forget the disturbing nightmare Guybrush has after falling out of the tree. Other than the characters, the entire world gets a bloodred tint, Guybrush's parents show up only to turn into skeletons which sing a song that is actually a hint for a later puzzle, and finally LeChuck himself showing up, transforming into Guybrush from the first game and spraying him with the voodoo root beer.
Finding the skeletons of Guybrush's parents in the Waiting Area of the maintenance tunnels under Big Whoop
Guybrush does one of the most creepy and brutal things in the series himself: He rips off LeChuck's leg
Largo catching you in his room is pretty scary if you don't use the voodoo doll in time. This is echoed in the last part of the game when you have to do the same for LeChuck, except that Guybrush doesn't give you a warning before he bursts into the room..
Rum Rogers Jr's Basement, in which there is the skeleton of his father in the bath. This is made funny by the fact there's a toaster in the bath (making it anachronistic) but the presence of it is still scary.
The game has one of the darkest sections of the entire series on Blood Island, when you have to investigate the Goodsoup family, especially when you go to the crypt. Blood Island's permanent night has quite a tone of foreboding to it.
The entire Goodsoup Resort, though entirely harmless, has a bit of a creepy air about it, especially when you go upstairs. It only gets creepier when you enter the furthest room down the hall and the music vanishes. When you pull the bed down from the wall, Guybrush finds a mostly decayed corpse. And then it gets WORSE when you lever the bed back up later and the skeleton shoots through the wall and parachutes down into the crypt, visible through the window, laughing hysterically all the way down. It turns into a hilariously romantic cutscene moments later, but Guybrush still ends up back in the crypt afterward.
LeChuck's resurrection. The screaming of his two unfortunate victims, and later, the pirates who ride his coaster.
Though never shown, it's mentioned that between the events of Revenge and Curse, Guybrush underwent horrible torture at the hands of LeChuck. When he finds himself back at the Carnival of the Damned, where it all took place, a hint of despair enters the normally cheery Guybrush's voice.
The Mysts o'Tyme Marshe, in which you occasionally come across your future self. It's extremely creepy if not outright scary, especially as it relies on memory to complete it (if you get it wrong you cause a time paradox and have to restart this section).
Humorously lampshaded when you melt down the Pyrite Parrot, whose voice slows down, and Guybrush comments that that's a little disturbing.
Less humorous is the Marquis De Singe, who performs amputations on all his patients, no matter how minor the injury, and when Morgan LeFlay breaks his finger, it heals.
He also suffers from a horrible Family-Unfriendly Death, as his turban that is supplying him with his Healing Factor is eaten by moths, causing his wounds to catch up with him. He wearily chases after Guybrush's animated severed hand, which he had been using to fuel his healing, only for the hand to push him into his own wind machine, causing his matter to be broken down and scattered across the seas. His scream cuts off abruptly as the machine spits out his remains like a woodchipper.
Another death, this time belonging to Guybrush himself! While getting run through with a cutlass isn't the most violent death, especially due to the Bloodless Carnage, Guybrush's inability to die due to him being in a LucasArts game, or in this case a Telltale game, has been a small Running Gag throughout the series, so the fact that it actually happened is that much more painful.
The giant manatees, especially considering that they shouldn't even be scary at all.
The absolutely brutal beatdown LeChuck gives Guybrush near the end. He even goes as far as keelhauling Guybrush! (For those who don't know, keelhauling involves tossing a person overboard and dragging them along the underside of the ship by pulling them back out from the other side with a rope.)
LeChuck's fate in The Stinger. All that's left of him is a bottle of green goo with his skull belt buckle floating in it, and he is screaming in agony.
Chapter 4: D'Oro lost an eye offscreen, and apparently he thinks that eyepatches are out of style, so instead he leaves the gaping hole exposed.
Chapter 3: Bugeye's trump card of a scary face involves sticking one of his eyeballs out of his mouth. When challenged to "top that", Guybrush asks "Are you suggesting that I pull out BOTH my eyes??"
The eye-related horror continues shortly after when Guybrush breaks into his first fit of the Pox since he lost his hand. He doubles over, holding his head, and when he glares up at Bugeye, the whites of his eyes are blood-red, and the normally friendly blue is a demonic yellow. His eyelids twitch constantly and the Pox spreads visibly across his skin, and though the eye color wears off, his skin remains spotted with green until you find La Esponja.