This is based on opinion. Please don't list it on a work's trope example list.
Nightmare Fuel / My Little Pony 'n Friends
In "The End of Flutter Valley", we're treated to a glimpse of what the valley would look like if the witches succeed. It's not pretty.
When the bees cover the Flutter Ponies in honey, Sting expresses concern that they may be drowned in it.
Later, a fire breaks out in Bumbleland and the trapped Flutter Ponies can sense the honeycomb getting hotter until the walls are practically melting. Part 5 closes on them screaming for help.
At one point in part 8, literally every heroic character is either captured or presumed dead.
The opening to "Return to Tambelon", with the unicorns disappearing after winking out with no explanation as to why. Grogar himself is pretty nasty too, at one point shooting lightning at the ponies with intent to kill and being nigh-unstoppable until the finale.
"Fugitive Flowers" has the Flories, plant-like creatures who exploit the fact that they look innocent to trick the Ponies into defeating their enemies, the benevolent yet fierce-looking Crabnasties. They then suck the life out of the soil from Posey's garden, killing everything around themselves and growing into giant, ugly monsters who plan to drink the life from the entire valley.
Not to mention they manage to hold Masquerade hostage at one point by tying her up in their vines, with a sinister, "She won't be going anywhere!"
Erebus's plan in "Bright Lights" and the effects a lack of shadows has on the ponies.
The shadow-less ponies in "Bright Lights" advancing on Knight Shade. The angle they're shot at makes them look like zombies.
Princess Porcina turning Ponyland and its inhabitants into glass. She's not even aware of what she's done at first, saying that when she looked through her mirror the ponies didn't seem real.
Shruck threatens to do the same to Megan and the remaining heroes—and then shatter them one by one.
In "The Magic Coins", Baby Lickety-Split, petulant over rain canceling the ponies' picnic, unknowingly steps on a magic coin as she wishes for it to never rain again. Cut to weeks later, where everything is drying up under the sun and the Baby Sea Ponies are dying of dehydration. And then a fire starts...
Niblic the troll threatening Megan and the other ponies to plummet to their deaths via drawbridge.
In "Woe is Me", the ponies come across Woebegone, a hobo whose bad luck affects him and those around him. In the end of part 1, he accidentally causes a mudslide where Baby Shady almost drowns.
"Baby, It's Cold Outside" has King Charlatan, an evil emperor penguin. While his design is cartoony, his plan isn't. He plans to freeze all of Ponyland, and when his son tries to stop him, he freezes him solid.
"Somnambula", which seems to owe something to The Lorelei Signal. Somnambula is a witch who lets every pony live their dream, with everything they wanted, even as she saps their energy, accelerating their aging so fast that in a matter of hours they're really long in the tooth.
It's a Near Villain Victory, too; all the adults would have died had not the bird taken a chance to flee and then break the hypnosis and had not Slugger seen his beloved in this state and gotten his Heroic Second Wind.
"Crunch the Rockdog," an emotionless stone bulldog who turned ponies and Bushwoolies to stone and was only defeated through the use of the literal heart of a living mountain.
Even worse, when the ponies removed the heart, the once friendly mountain began to attack them as he'd lost his feelings without it.
"Would Be Dragonslayer" has a squire trying to kill Spike in order to become a full-fledged knight.
"The Golden Horseshoes" has Mimic falling ill for what initially appears to be no reason. Her symptoms include weakness, a pale complexion, no appetite, and even the loss of the twinkle in her eye.
The living chair monster in "The Ghost of Paradise Estate."
Not to mention the door to the nursery.
"The Quest of the Princess Ponies" has Lavan, a lava demon who turns himself into a crystal being with the Princess Ponies' wands and unbalances the magic of Ponyland. When he's defeated, he explodes.
"The Prince and the Ponies" has the First Tooth Baby Ponies jealously wishing horrible fates on the Newborn Twins, including being struck by lightning and kept as someone's pet. The latter happens, with the Duchess's daughter abusing the ponies and keeping them in chains.
They also form some great goblin-like Nightmare Faces during the musical number they sing.