What are they? I consider the most difficult villain to be Iron Legacy, but Miss Information took the longest to beat. BUT I ragequit the most against Chairman, Cosmitron has the toughest challenge mode, Proletariat has a nightmare ultimate mode, PR is the worst team villain on standard, Oblivaeon is just insane, Gloom Spite is the most difficult variant, and I don’t even know where to start with Wager Master.
All that’s just my opinion. So what’s yours?
If we’re talking Enhanced Edition, I’d probably say Dawn. Matriarch and Infinitor are also up there. Oh, and Spite. And probably Chairman.
For Definitive Edition . . .honestly, none that I’ve played against seem especially harder then the others. I’ve only played all the base Villains in the Core Set save for GW Voss, though; no Events or RCR Villains.
I think there are two standards here (at least for EE).
A lot of villains are extremely difficult until you learn their trick, at which point they’re very easy with the right composition. The Matriarch, the Chairman, and Iron Legacy are all absolutely brutal, but they’ve also all got ways to shut them down. I think if you’re just going random, Iron Legacy probably remains the hardest. He’s just a very hard, very fast hitter and you can’t safely whittle him down or he flips and wrecks you. The tricks to deal with him easily are a lot harder to acquire. (Agent of Gloom Spite is another example, because the tricks to beat him involve deliberately not engaging with several key parts of his deck in favor of focus fire.)
Once you know everyone’s tricks, I’d say the hardest villain is Kaargra Warfang, because she’s a moderately difficult challenge that’s almost impossible to build good tricks against. There’s just a bunch of routine ways that she can mess you up almost indefinitely, and the Favor Track means you can’t use some of the normal puzzle boss strategies.
Challenge Mode upends all of that, because a lot of the challenge modes do extremely weird things to decks. Challenge Mode Ambuscade is incredibly swingy, with the potential to win before the players get a turn, or to stumble along and die in three turns. Challenge Mode Cosmic Omnitron is absolutely awful. Challenge Mode Chokepoint is either a nightmare or a pushover depending on your equipment layout. On the other hand, Challenge Mode Chairman isn’t too bad, and Challenge Mode Matriarch might never really come up.
Agree with FrivYeti, it really depends on your team composition (again, at least for EE). That said, if we’re neutral on composition (no hard counters like Haka’s Savage Mana vs Chairman, but no weaknesses like Wraith vs Shu of the Enead)… I would say that Miss Information and Wager Master have been the most causes of frustration, for me. “I’m not loading them up if I’m not trying to unlock a variant” are a good summary of those two…
Now, been playing through some DE stuff ever since they came, and I have to say that Dawn is so far the toughest of the batch, but I haven’t played through a fight against the Organization, yet. Omnitron is probably the most frustrating from a bookkeeping perspective. Apex is my new favorite fun brawl.
We actually played through our first Organization fight two nights ago. Base Expatriette, Mentor Nightmist, FA Argent Adept, Detective Wraith. The only one chosen based on “gaming the system” was Wraith, as I was last choice, and wanted to help Argent set up faster. A lot of mutual support, with most of it aimed at Argent for setup, and Expatriette for extra damage/extra power use. We did get down to single digits HP at one point, but honestly the most difficult part was keeping track of what activated each turn.
Expatriette murdered so many gangsters. Scary what she can do now in DE.
A couple friends and I recently did a full campaign of all 30 of the events and critical events in DE. We did not use advanced mode and we did use collections, which did make things much easier.
The only 2 that we lost to were Grand Warlord Voss and Sunrise Citizen Dawn. Normal Terrorform came close, knocking out everyone but Absolute Zero, who was able to solo his flip side thanks to help from Magmaria.
I’d say Sunrise Dawn is probably the hardest villain in DE so far. She’s very difficult to stabilize into something manageable. Her ongoings are all bad, she’s guaranteed to have one every turn, and killing problem citizens is pretty futile since she’ll likely play Return with the Dawn and bring them all back. Plus she still has That Card and 120 health; she’s brutal!
Thanks for that DE input, but I don’t own it, just EE.
I don’t have mini packs 1, 2, 4, void guard or oblivaeon, but the classic villain that took me the longest was Iron Legacy. Hardest team villain was PR
Honestly, in no world is Ambuscade difficult, so not having mini-pack 1 doesn’t change much.
He can be with challenge and ultimate rules in EE.
I am excited to try challenge-it looks neat.
“Difficult” no, but he can be real annoying if the deck keeps cloaking him…
I have played ambuscade with a friend in physical. Wasn’t that hard
I love playing team mode with Plague Rat! he’s great for taking out the other villains! 
Baron Blade in team mode has generally been our toughest opponent, as he is frequently working with the other villains in ways that makes him scale far harder against larger groups of heroes.
If you take Blade out early, the game goes much more smoothly. I usually play him with 3. In many of my games, especially on advanced, Plague Rats damage to villains gets reduced. I beat both though! 
I have repeatedly been ripped to shreds by the chairman and out of 9 games the closest I have gotten is barely killing the operative.
EE or DE?
I would only play Wager Master if I was unlocking a variant or its a weekly one-shot
Try Visionary, Bunker, or, if you’re really risking it all, Tempest.