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Vincent Van Gore
Vincent Van Gore is the creator of the ghosts in the original Luigi's Mansion. He's essentially a wild artist used by King Boo to create spooky fiends for Luigi to confront. However, he's ultimately almost a non-entity in the game, only appearing toward the end as a boss before King Boo. He's a good boss fight, but not a particularly deep villain.
Bowser Jr. is a strange figure. He's ultimately one of the main antagonists of Super Mario Sunshine -- until Bowser shows up. Bowser Jr. is Bowser's child, believing Peach to be his mom. Bowser Jr. is ultimately a less-interesting counterpart to the Koopalings. While not bad, he's also nothing particularly special.
Elder Princess Shroob
Elder Princess Shroob is an alien invader who tries to take over the Mushroom Kingdom. She's the main antagonist of the game, being the true leader of the Shroob aliens. Her design is grotesque and monstrous, but she's ultimately absent for most of the game, due to being, supposedly, sealed away early on in the game. She's a good boss, but not a particularly memorable one.
Wart is the main antagonist of the second Super Mario Bros game, and, immediately afterwards, they went right back to Bowser and never looked back. Wart is a gaudy toad whose reputation has become more favourable over the years. However, none of this changes that he's essentially a less-interesting Bowser. It's telling that his servant Birdo has proven more popular than him!
Kamek is a loyal soldier under Bowser, serving the Koopa King since his infancy. He's a magic figure who can turn common enemies into threats great enough to challenge the entire Yoshi race. However, Kamek is ultimately a relatively minor character. He's iconic, but he's also just a servant of Bowser.
The Koopalings served as fairly iconic bosses in Super Mario Bros 3, being Bowser's many children. Unlike Bowswer Jr., every individual member of the Koopalings is distinct in both design and personality, standing out from Bowser. Of special note is Ludwig, who believes himself to be the leader of the Koopalings -- despite how no one actually held a vote to determine who led them.
Master Hand and Crazy Hand
Master Hand and Crazy Hand are, technically, not Mario villains. Rather, they're the reoccurring villains in the Super Smash Bros series. While the franchise has even greater villains above the Hands, they remain the most iconic forces in the franchise, being the final boss in Arcade Mode. As such, Mario and friends have beaten these cosmic crossover antagonists time and time again.
Hellen Gravely is the secondary antagonist in Luigi's Mansion 3, owning the Hotel that Luigi and friends end up staying in. She's a very fabulous ghost with a love of make-up. She idolizes King Boo, helping free him and allowing him to enact his second attempt at revenge against Luigi. Personality-wise, she's hard to peg. However, her sheer appearance leaves her as one of hte most memorable of the Luigi's Mansion series' ghosts.
Fawful is one of the main antagonists of the Mario & Luigi series. He's essentially a crazy, unstable mechanical genius whose schemes end up drawing the attention of Mario, Luigi, and even Bowser. He's definitely a wild and crazy figure who is memorable for just his sheer disaster energy.
The Shadow Queen
The Shadow Queen feels more like a Sailor Moon villain than a Mario Bros one. This ancient evil queen was sealed away a thousand years before the events of Paper Mario and the Thousand Year Door. She's unleashed by Big Bad Wannabe Grodus, only to be immediately betrayed. Shadow Queen is memorable for possessing Peach and using her as her personal avatar to enact her dark war against society. She remains one of the most fearsome villains Mario has ever faced.
Waluigi is the unsung hero of the Mario franchise. While Waluigi has never been a main antagonist outside of the sports games, he's become an icon all the same. Waluigi feels so out of place in the Mario world, since his rivalry with Luigi isn't as established as Wario's rivalry with Mario. However, Waluigi remains as this strange, awkward fixture. And he's incredible.
King Boo is the primary antagonist of the Luigi's Mansion franchise, while also being a major villain throughout the Mario franchise as the leader of the Boos. King Boo is one of the few villains to have successfully defeated Mario and friends -- more than once, actually. Despite his silly appearance, he's managed to outdo Bowser, despite sometimes aligning his forces with Bowser. The only person who can stop him is, apparently, Luigi.
Wario has since become more of his own character, but he started as a primary antagonist back in Super Mario Land 2. He's a greedy monster who just wants to horde everything for himself. While he's gained popularity on his own terms in his own games, he's still Mario's ultimate foil, being the exact counterpart and rival Mario needed. It is fascinating seeing how this character has evolved separate from Mario, much like....
Donkey Kong was Mario's first antagonist -- or, at least, Kranky Kong, Donkey Kong's grandfather. The world of Donkey Kong is complicated. In many ways, Mario is Donkey Kong's enemy, since Mario tortured Kranky Kong as his pet then kidnapped Kranky Kong, leading Donkey Kong Jr to become a hero to save his father. Kranky Kong's grandson is the most famous Donkey Kong, but even then, the line of gorillas is diametrically opposed to Mario. But, over time, they've gained their own unique lore.
Are you surprised? No, really, are you? Bowser is obviously the best Mario villain. There was no debate here who would be on top. Did you think I was going to pull a fast one on you and put Waluigi here or something? No. Bowser is just the best. He's reinvented himself from game to game, pulling out new and wild schemes. I don't need to justify putting Bowser this high up, do I? He IS Mario's ultimate villain.