Coming to think about it, the Shroobs had to be the most dark Mario antagonists so far.
The Shroobs were devoid of any sense of morality. Though they were trying to survive their solutions to taking over the other planets are somewhat barbaric. Vim is basically the souls of the Toads which is harvested to power the Shroob weapons such as the UFOs. To make the Shroobs even more sick in the head, their second in command, Princess Shroob carries a martini glass with Vim in it which she drinks from a lot. She drinks liquid lifeforce, that is sick and wrong. Queen Shroob is also shown to be an absolute beast that really shows no concern for her subjects, just like her sister. She smashes a minion's ship to pieces, killing the pilot, all for a simple task such as bringing her a Vim glass, and in her second phase, she shoots them down so they crash into the Mario Bros. The Shroobs as a whole were a messed up and disturbing evil empire. It's hard to believe a normally purely comical series such as Mario & Luigi got away with this, because a plot that is this serious is mostly in the Paper Mario series (not counting Sticker Star). Like Count Bleck trying to erase entire worlds just because he thinks after Tympani's supposed death made nothing else matter anymore in Super Paper Mario. Sure Bowser's Inside Story got a bit dark with Fawful trying to awaken an apocalyptic semi-sentient artifact and absorb it's power so he would shroud the world in darkness for eternity, but that is nowhere near close to the barbaric and inhumane atrocities that the Shroobs caused throughout PiT.