Batman: The Animated Series has The Joker, as usual per being the Arch-Enemy of Batman. He's the most recurring threat out of all the villains, although in terms of scale, he doesn't quite match it with his usual penchant for chaotic humor. Until later on in Justice League, he gets his hands on Ace, and a government satellite.
The series featured an ongoing arc of Superman's struggle with the Galactic Conqueror Darkseid, the ruler of Apokolips, and his increasingly daring designs on Earth. By contrast, Lex Luthor's schemes of being a Corrupt Corporate Executivehiding in the public were almost purely episodic in nature. Darkseid only first appeared briefly at the end of the first season, however, with the later seasons being what intensified his conflict, eventually culminating in brainwashing Superman, which damaged his reputation.
For much of Superman: The Animated Series and Justice League. Lex Luthor bankrolled individual supervillains, founded the Injustice Gang, provided secret funding to Cadmus, and eventually usurped the leadership of the Secret Society, motivated by hatred of Superman and desire to be seen as the hero to everyone else.
Batman Beyond had Derek Powers for its first season, being responsible for the murder of Terry's father, which drives Terry to become the new Batman and his corporate takeover of Wayne-Powers acting as the overarching conflict of the season, eventually becoming a supervillain himself as Blight. However, after his defeat in the season finale, the show's following seasons would lack a main villain.
Static Shock had Ebon, a criminal mutated by the Quantum Vapor who acts as the head of the "The Meta-Breed" Gang, a group of Bang Babies acting under his behalf that are frequently at odds with Static.
The Big Bad of the Cadmus Arc was constantly being teased. At first it seemed Amanda Waller was the mastermind, as the head of Cadmus who was trying to protect the world by aiming to counter the Justice League out of paranoia in response to Superman's earlier brainwashing and the brief visit by the Justice Lords, being revealed to have created Doomsday and the Ultimen and Task Force X as a result, then Lex Luthor, who reforged his Villain with Good Publicity act and manipulated her to make himself the hero at the cost of demonizing the actual heroes. It was finally revealed in the next-to-last episode to be Brainiac, who had reformed from within Lex, was manipulating his earlier actions in the name of restoring himself, and then enters into a Big Bad Duumvirate with him where they fuse together to fight the League.
There were several contenders for the title in the final season. At first it looked like Grodd, a sapient Killer Gorilla who originally created the Secret Society of Super-Villains and had recently expanded its roster to combat the larger Justice League roster, but when his evil scheme turned out to just be turning all peopleinto gorillas, he was deposed from leadership by Lex Luthor, who had recently joined this team. This later lead to an apparent Big Bad Duumvirate between Lex and the remnants of Brainiac's consciousness, seeking to fully restore Brainiac, which eventually leads to infighting among the Secret Society when Tala, a sorceress who worked for Cadmus before then joining the Secret Society out of favor to Grodd, frees the gorilla and turns a significant portion of the Society against Lex. In the end, though, when Lex forces Tala to use her magic to restore Brainiac, she instead chooses to revive Darkseid, who retakes power in Apokolips and becomes the Final Boss of the DCAU's ongoing saga by moving to conquer Earth, forcing the Society into an Enemy Mine with the League.
Films
Batman: Mask of the Phantasm: The Phantasm, who comes into conflict with Batman after systematically killing several of Gotham's mob bosses. It becomes a Big Bad Ensemble when Joker gets involved and tries to kill Phantasm himself.
Batman & Mr. Freeze: SubZero: Mr. Freeze, who kidnaps Barbara Gordon to use as an organ donor in order to save his wife's life.
Justice League vs. The Fatal Five: The Fatal Five, led by the Emerald Empress, who go back in time to blow up the sun in order to prevent the existence of superheroes in their future.
