Castle Bowser is a fusion between Bowser, his Koopa Clown Car and the Flower Kingdom Castle that happens when he grabs a hold of a Wonder Flower, and the fusion is very creepy, especially with his demented swirly eyes and dark Piranha Plant clouds that protect him from any outsiders while he plots to take over the entire universe.
Heck, how he wrecks havoc across the kingdom is pretty creepy. He converts all of the Popplins' houses into jail cells and has Bowser Jr. and Kamek cause their own chaos in several of the lands.
Bowser Jr. himself looks creepy during some of his battles, specifically adopting a similar color scheme to Dark Bowser and a creepy mask that happens to have sharp teeth itself.
His battle in Deep Magma Bog deserves a mention, considering that he turns the battlefield into a silhouette one during some of his phases, with the platforms growing really creepy orangish eyes which look very similar to the Eye of Sauron.
The Talking Flowers are usually friendly, but the ones in the Pyramid Wonder event in "The Desert Mystery" are the exception. You see reddish ones (keep in mind that red ones usually hang out in Badge stages and are encouraging) that will outright shout "Intruder!" as you enter the pyramid, with strong implications that they set up a trap as well. This is definitely something when most Talking Flowers are either friendly or just want to provide their own commentary and exposition.
The entirety of the level of Muncher Fields, which happens to be situated right in front of Castle Bowser in the world map. The atmosphere screams Bleak Level compared to everything that's been thrown at you so far - with rain being present, Bowser giving you an unsettling stare the whole time, and the creepy Fungi Mines music playing in the background. Thankfully it's a Breather Level compared to the two worlds it's situated between (Fungi Mines and Deep Magma Bog) but it's still really creepy, even by Mario standards.
The purple variant of poison in certain levels of Pipe-Rock Plateau looks dangerous, but at least it's a familiar sight from previous Mario games. The dark blue poison with bright green outlines in Fungi Mines, however, just looks wrong. It makes an unsettling sound as it pulsates, and in A Final Uncharted Area: Poison Ruins, the poison will start rising once you touch the Wonder Bud on the first bone raft. Before you enter the pipe that leads to the real exit, you can see the poison spontaneously fill up the shaft that you fell into at the beginning. The dark blue poison becomes even more menacing while you're free-falling in "Fungi Mines Special: Dangerous Donut Ride", as the poison will form into circles that must be dodged and walls that constantly shift back and forth and close in on the center.
You endure the gauntlet that Castle Bowser throws at you and you're ready to take him on. Bowser Jr. then activates a "super duper extra-cool Wonder" which backfires on him. This Wonder ends up being a distorted mass of everything that's been thrown in at you, with the music sounding very off and the Talking Flowers which appear have very unsettling voices compared to what you encountered beforehand.
playing in the background. Thankfully it's a Breather Level compared to the two worlds it's situated between (Fungi Mines and Deep Magma Bog) but it's still really creepy, even by Mario standards.