- Dark Awakening, Zombie Optimus Prime and his Heroic Sacrifice that involved bits of him being blown apart, he ends up without an eye and an arm before his death. The first act had Optimus Prime terrify young Daniel after the lights go out.
- Webworld, the Torkuli in general. They are not above lobotomisation to cure their patients of their insanity and speak in a Creepy Monotone. The fact that Cyclonus (who signed all those forms to have Galvatron committed) couldn't do anything to stop them from them implementing Maximal Intervention (by use of the Ayla, insects that have a symbiotic relationship with the planet) on his leader.
- The Energy Vampires (which other than the name, are pretty much a robotic version of the classic Romero zombie) from "Dweller in the Depths" are really creepy. They lose all their coloration, their eyes glow, and they advance slowly but utterly implacably.
- The Hate Plague from "Return of Optimus Prime" spreads across the entire planet, so everyone has to see everyone they love go insane and start attacking them, knowing that just a touch means they will lose their minds as well...
- Galvatron, the first one, especially after he goes completely insane. As his earliest appearance demonstrates, he can shrug off enough firepower to level a whole city.
- The Demons, Primus' first failed creations, are all kinds of unsettling.
- Shockwave's first appearance in the comics, casually shooting every Autobot present. No warning, no announcement, he just shows up almost completely out of the blue and BLAM!
- The corrupted Matrix. Especially once it starts taking over Thunderwing, growing into a large demonic shade...
- Its reappearance in Regenesis One is so much worse, though.
- Rhythms of Darkness. All of it. Unicron has won. Cybertron is gone. The Matrix is gone. The war is long over, and the Decepticons have won, and taken over America. The rest of the world is planning to nuke the Decepticons, unaware it'll just make them stronger...
- Buzzsaw. True, he's a walking version of The Worf Effect, but his method of fighting is disturbing; slicing his enemies to pieces with his beak. And it takes fifty issues for him to go down for good.