This is based on opinion. Please don't list it on a work's trope example list.
Nightmare Fuel / Transformers Animated
Blackarachnia, from the Transformation Trauma of her backstory on up through her vampire-style fangs and hissing. Let's not even start on nearly causing all of Detroit's organic life to wither and die when she tried to use the key. She looks even worse with her helmet off.
Lockdown in general. Removing parts from still-conscious bots with surgical precision so he can use them as his own later on or for trophies is just plain disturbing.
Don't forget that his debut episode established that Transformers do feel pain.
Lugnut nearly getting eaten by AllSpark-charged nanobots and cut in half.
Wreck-Gar, for a second there on the barge. [Usual cheerful voice all the way through] "I am Wreck-Gar! I dare to be stupid! [Eyes turn bright red] I will destroy the whole city!" It was... very different from the other times he's casually lapped up other people's suggestions. Best seen in context.
Wasp's speech pattern and appearance were a shoutout to the loveable but luckless Waspinator from Beast Wars — except that Waspinator wasn't originally a mentally normal if slightly jerkish person. The years that Wasp spent imprisoned for treachery against the Autobots apparently were horrible enough to completely warp his mind, and this is from the good guys. Then he tries to get his payback by switching places with Bumblebee, Face/Off style. Not only do you get to watch Bumblebee's horror at being trapped in the likeness of the enemy, you get to watch Wasp remove his face. But much worse was Wasp's creepy behavior through the whole scene, with that childlike, playful, way-too-calm voice he uses as he's doing it.
And then in a later episode, Wasp is transformed into the hulking technoorganic Waspinator. It's mercifully offscreen, but you can hear his screams and see his hand pounding at the glass of the chamber door. Later in the episode, we have another Beast WarsShout-Out turn disturbing. Yes, he does say ''Waspinator has planzzzz" while trying to pull himself back together, but he's sparking and leaking everywhere as he says in the most menacing of buzzing voices, hilarious dismemberment becomes horrific.
You gotta hand it to the writers, they took the Chew Toy of Beast Wars and played him as shockingly straight as possible.
Blurr got one of the most brutal moments in the show when he gets caught between two Advancing Walls Of Doom and gets crushed into a metal cube.
Derrick Wyatt stated on his blog that Blurr would have, through a series of distractions, avoided his fate in the incinerator. He would have survived the rest of the series as a cube, unable to move on his own. Also, an animation detail that got left out? His still beating spark.
Sari, after learning of her semi-robotic nature decides to use the Allspark Key to take a level in badass. At first it works... but then she completely loses control of herself and comes all too close to graphically killing her best friend Bumblebee when she accidently impales him.
Blitzwing, who is so funny that he doesn't seem scary at all... until you realize that he's a completely deranged, out of control, overpowered war machine, who gets double the weapons when he's at his craziest. And let's not get started on the way he laughs. Also, let's not ask what "servo salad" is made of.
Protoforms are like baby or fetal Transformers. Starscream's clones require protoforms to create. There were what was likely dozen of clones in that room. Megatron destroyed them all.
And, speaking of Starscream's clones, let's not forget that at least the first two were for all intents and purposes suicide bombers. Made with stolen 'babies'. Yeah.
Watch "Human Error". Awesome, yes? Well, look at Bumblebee again. He looks even younger than Bulkhead (who looks to be well into high-school). He can't be any older than Sari. Soundwave had to give them all forms that they (and we) completely bought into - Ratchet even had a scar to represent his broken chevron. So in Cybertronian optics, Bumblebee is at an age where he should be considering a paper route an intense job, but he applied for the Elite Guard. It's really quite disturbing in some ways, when you think about how young he looks in that episode.
Soundwave in that episode was pretty damned scary.
How far has this page gone without us having mentioned Megatron himself? Affably Evil, yes, but also pretty scary at times. There's that Creepy Monotone, for starters, and the fact that he's The Dreaded, and proves himself to be incredibly dangerous. Strangely, when he's a disembodied head in Season 1, he's even scarier. There's the fact that all the technology in Detroit is reverse-engineered from him so that he has access to all the machines around him, which is proper Paranoia Fuel; he tries to kill the Autobots in "Home is where the Spark Is", and his face appears on an ATM in "Nanosec".
The Jettwins creation into what they are now. Before they were repair bots before they got caught in an explosion. The only reason they survived was that Ultra Magnus wanted expendable bots that they could upload Starscream's CNA into. Ultra Magnus probably knew that something was going to go wrong and the twins would have to be terminated.