With little over 48 hours between games and a squad seriously depleted by injuries, the DW Stadium performance exceeded O'Neill's wildest expectations.
Craig Gardner gave the visitors the lead with a stunning free-kick right at the end of the first half before James McClean netted his first Sunderland goal.
Hugo Rodallega pulled one back for Wigan but their hopes were quickly extinguished by a Stephane Sessegnon strike and David Vaughan added a fourth late on.
O'Neill said: "I thought it was the best result since I arrived. Manchester City was a great win for us, to score in the last minute, it seemed as if we'd played three games, particularly in the last 20 minutes of the game.
"We've had a day less to prepare (than Wigan). I accept the fact the adrenaline's still flying with us but to come here and win today [Tuesday] was a fantastic performance. I just don't know where they got the energy and will to keep going from."
The final scoreline looked distinctly unlikely in a first half that Wigan dominated, with Gardner clearing off the line from Steve Gohouri before David Jones and Ben Watson both hit the post in a crazy 10 seconds.
The key moment of the half came at the other end, though, when deep in injury time and with rain swirling around the DW Stadium, Nicklas Bendtner won a debatable free-kick and Gardner sent a swerving 30-yard shot into the top corner.
The visitors doubled their lead 10 minutes into the second half, 22-year-old McClean heading in at the second opportunity, before Rodallega gave Wigan hope with a deflected shot for his first goal of the season seven minutes later.
But the game was killed off in the 73rd minute when Bendtner crossed for Sessegnon and 10 minutes from time Vaughan added some gloss with a stunning strike from 20 yards.
Sunderland, limited by their injury troubles, played largely on the counter-attack and O'Neill conceded they benefited from some moments of fortune.
He said: "I thought Wigan controlled the game early on but they're playing at home and they're going to get the ball. We were always dangerous on the counter-attack and could have scored a couple of goals ourselves in the first half.
"But, having said that, I do accept we rode our luck a little bit. We got the goal just before half-time and I thought our second-half performance was fantastic."
The victory took Sunderland's haul since O'Neill took charge six games ago to 13 and left Wigan rooted in the bottom three.
The Latics' 2-1 victory at the Stadium of Light in November was Steve Bruce's last in charge of the Black Cats and also kicked off a more promising run of results that was brought to a crashing halt on Tuesday night.
Manager Roberto Martinez was once again left bemoaning the rough end of a refereeing decision after Mike Dean ruled Watson had felled Bendtner for Gardner's opener.
The Spaniard said: "The first goal was going to be vital. In the situation that Sunderland were, they had an outstanding result two days ago and we knew we were going to play a side with great confidence but probably who were going to sit deep and defend.
"It was going to be a matter of creating enough chances to open them up and score the first goal. I was delighted that we were patient in the first half, we opened them up two or three times and hit the post twice.
"They were key moments and then Sunderland take the lead with a world-class strike from a free-kick that was non-existent and from then on it became a very hard job. We wanted to open the game, we took risks and Sunderland took advantage on the counter."