This is based on opinion. Please don't list it on a work's trope example list.
Nightmare Fuel / Equestrylvania
Death. Everything about this guy is terrifying: the fact that his face drives ponies to madness, the fact that his voice is made up of the voices of every person he has ever killed, and now that he’s collecting ponies’ cutie marks...
Plus, we know that he was always one of the hardest bosses in each Castlevania game he was featured in, and now we know he really has it in for the ponies. This isn’t gonna be an easy fight for them…
When Death gets angry, his skull shifts and contorts in spasms. Even Actrise is freaked out by this.
A lot of the Mood Whiplash. First, the author takes time to flesh out characters, throws in some heartwarming scenes, some comedy, and everything is well and g SURPRISE DEAD CHILDREN AND A NURSE COMMITTED SUICIDE
Twilight's dream sequences delve into this. In particular, the constant mention of the "Bloody Tears" is the single, still image she has of the bite marks on Shining Armor's neck.
Marble. Poisons children, only to go out of her way to intimidate the heroes in any and every way that she can. Her calm description of the process of killing something is chilling.
Worse, it wasn't even really Marble. She was possessed by Actrise... who undoes the possession by crawling out of Marble's mouth, leaving her lying on the ground, feeling sickened and violated.
The Reveal as to why Shatterstorm has such issues with mares: he's been repeatedly abused, emotionally and sexually, by various mares. Including his own mother. The epilogue of Book 1 involves him taking a Shower of Angst after being taunted about his past by Actrise-as-Marble in previous two chapters.
Roseluck's behavior has become more and more disturbing. At first, she's just kind of kooky. Now? She's speaking in a Creepy Monotone, distrusting of others, talking to herself, lashing out violently...
Babs getting chased through the streets of Manehattan by one of Dracula's monsters, and desperately having to hide in a literal hole in a wall after being cornered in an alley.
The remaining Royal Guards have become He Who Fights Monsters, beating and torturing accused traitors to death and mutilating the corpses.
And then it gets worse when it turns out that Rose Blade and his followers are actually working for Dracula, and Rose Blade gives Rainbow Dash the Sadistic Choice of either becoming his sex slave or him killing Shatterstorm.