The greatest villains in DC Animation Universe. Practically includes TAS (+ The new Batman Adventures) and movies that belong them.

#20 Leader of Terrorist group called Multigon with the intention of spreading poisons and bioweapons in Gotham and got into a conflict with #11. A worthy opponent but still she feels Lady Shiva knock-off.
#19 Evil AI, a machine that replaces humans with robots. Scifi horror at its best with a combination of Terminator, Invasion of the Body Snatchers and 2001 Space Odyssey.
#18 Corrupt businessman who tries to take over Wayne Enterprises and was behind the creation of #9. Perhaps most remembered for his appearance in The Dark Knight Rises film.
#17 Female anti-batman and a variant of Batman foe Reaper. Main villain in animation movie Mask of the Phantasm.
#16 A sideshow freak and strong-armed criminal who almost got Batman with a big rock.
#15 A schizophrenic man with his creepy gangster dummy. Episode "Double Talk" is pure Hitchcockian psychological thriller, probably the best episode in "The New Batman Adventures".
#14 The underlying criminal boss in Bronze Age but more important role in the series who was behind the creation of #4 instead of Sal Maroni and hired #12 to take out Batman.
#13 Leader of League of Assassins, "the Demon". His episodes in TAS are quite faithful to the comics.
#12 Main hit-man and strong opponent for Batman in TAS. Episode "Bane" is one of the best adaptations of the character since "Knightfall" and main villain in animation movie "Mystery of the Batwoman".
#11 Animal activist and a cat burglar. Especially later episodes in the series she has probably the best villain era since 70s. Short film called "Chase Me" is perfect cat and mouse play between her and Batman.
#10 He has only 3 episodes which is quite few compared to other main villains but those are great especially virtual reality episode "What Is Reality?" is immemorial.
#9 Matt Hagen, A tragic actor. #18 turns him into a mud creature in touching monster episode. Actually the origin story is more Basil Karlo's than Hagen's, Silver Age version of the character.
#8 Gotham's boogeyman, master of fear toxins which made Batman almost psychopathic in "Never Fear" and the episodes in TAS had a strong influence on "Nolan verse" overall.
#7 Criminal mastermind in Gotham and one of main Batman villains in TAS who almost got him several times even when Batman was blind as a bat.
#6 The best version of the character so far while in comics he is often a little creepy killer who lives in fairytales. Especially "Perchance to Dream" is one of the greatest episodes in TAS.
#5 An eco-terrorist, one of main villains in TAS with man-eating plants, tree monsters and fatal kiss. Later friend and partner of #2.
#4 An ex-lawyer with latent mental health issues who was pushed "over the edge" by #14 in psychological thriller-like episode(s) "Two-Face". Later one of the main villains in TAS.
#3 Generic ice themed mad scientist whom TAS made as the most tragic supervillain not only Batman's but overall in the fiction. Main villain in animation movie "SubZero" with his beloved sleeping beauty and pet polar bears.
#2 A prison psychiatrist whom #1 corrupted into his own sidekick. This cheerful acrobatic villain quickly became fan favorite in TAS and later even overused in the comics and no wonder. Everything essential about her was already developed in TAS; the origin, the problematic relationship with mistah J, her friendship with #5 and so on.

HM1: Dr Jekyll and mr Hyde style villain, later his beloved turned into monster herself too.
HM2: Woman in spoiled little girl's body might sound silly but it still works.
HM3: Daughter of #13 and his faithful helper. This version is quite faithful to the source material too but still a bit more evil than the bronze age version.
HM4: Time themed enemy terrorizing Gotham. More of Green Arrow and Teen Titans foe but Batman's one too since Batman 60s show.
HM5: A ninja from the past that comes back to haunt Batman.

#1 Clown prince of crimes. Placing him as number one starts to feel boring but it has reasons. 1) there is no #2 without him 2) The highest number of appearances makes him main villain 3) Adaptations of iconic stories like clown fish 4) This is not only TAS but DCAU overall, he made Tim Drake as his successor in "Return of the Joker"-movie and 5) great voice performances, thanks Hamill.