Main trio

Hiro Miyamoto

Big "NO!": Done when he's first sent back in time with the Daikatana.
Meaningful Name: He's a sword-wielding hero, and shares a name with the legendary Japanese swordsman Miyamoto Musashi.
Not Even Bothering with the Accent: While Kage and Mikiko have over-the-top Japanese accents, J. Paul Slavens voices Hiro Miyamoto with an American accent.

Mikiko Ebihara

Disney Villain Death: Mikiko falls in a pit of lava at the end of the Game Boy Color version as Hiro dodges her attack. Superfly also falls trying to save her.
Evil All Along: Reveals her true colors when she kills Superfly. Also, she tries to seize the Daikatana for herself at the end of the game.
He Who Fights Monsters: Subverted. After killing Superfly, Mikiko reveals that the Ebiharas were a bunch of bandits and cutthroats who wanted the Daikatana for their own selfish ends, just like the Mishimas.
Post-Final Boss: Attacks Hiro by stealing the Daikatana at the last minute after Kage's defeat.
Walking Spoiler: If you couldn't tell by all the whited out text above.

Superfly Johnson

Black Dude Dies First: Although he's not the first victim in general, Superfly is the first one of the trio (Hiro, Mikiko and him) to die in the final level. Averted in the GBC version, where he's simply knocked out first and instead dies right after Mikiko.
Inelegant Blubbering: Every time Hiro dies (no pun intended), Superfly starts to bawl, sob and cry loudly and, sometimes, he lets out a Big "NO!".
Soul Brotha: Just the fact that his name was taken straight from a Blaxploitation movie should already make him qualify. According to this article by one of the game's original writers, the character's name was originally Superfly Williams - a tribute to the blaxploitation series and Jim Kelly's character from Enter the Dragon. Initially conceived as a French character named after the few surviving cultural documents existing in the post-apocalyptic future, the end of the game would have seen him learn his true identity. After that writer left, the laziest possible route was taken.
Taken for Granite: He gets turned to stone by the medusa after the trio gets separated in 1200 BC. He gets better after you kill it.

Antagonists

Kage Mishima

Badass in a Nice Suit: In the N64 version, Kage Mishima sports this look instead of the elaborate samurai armor from the PC version.
Big Bad: He's the person responsible for the state of the world in 2455 and the person Hiro must defeat to set things right.
Tin Tyrant: In the PC version, he wears samurai armor.
Villain Has a Point: He warns Hiro to not trust one of his companions. He was right and Mikiko lampshades it.

Toshiro Ebihara

Small Role, Big Impact: Toshiro Ebihara. He doesn't survive the intro, but is the one who gets the whole game rolling by convincing Hiro to search for his daughter Mikiko and the Daikatana.

Usagi Miyamoto

Driven to Suicide: After wrestling with Inshiro Ebihara, Hiro's ancestor Usagi was mortally wounded and then, he threw himself along with the Daikatana in the Fujiyama's volcano.