Villa beat the mighty Ajax 2-1, thanks to goals from captain fantastic Martin Laursen and the ever excellent Gareth Barry on Wednesday night.
There was an almighty buzz at the old stadium and the team did not let the vocal home support down.
Ajax present more than formidable opposition for most sides yet, O'Neill's young outfit were not to be overawed.
Bristling with energy and hunger from the off, Villa frightened Marco Van Basten's team with their pace and hunger. Though, as always, it has to be O'Neill who takes the plaudits. Dealing with the massive blow that his monster target man John Carew was unavailable due to illness, his tactical reshuffle was nothing short of genius.
With the aforementioned Carew out, O'Neill drafted in James Milner to play wide right of a five man midfield, which also saw Barry resume his midfield duties.
Luke Young and Nicky Shorey both returned in the full back positions having been rested on Saturday and Carlos Cuellar was surprisingly chosen ahead of Curtis Davies partnering Laursen at the heart of the defence. O'Neill presumably favoured Cuellar due to his UEFA Cup experience.
Ajax started the game with some neat passing but it was Villa who struck first, when, on seven minutes the outstanding Laursen out jumped Ajax keeper Kenneth Vermeer to powerfully head the home side into an early lead and send Villa Park into raptures.
Initiative
It was from Villa's inevitable supply line too. The signing of Ashley Young's from Watford has proved to be just another stroke of genius from the Villa manager. The winger's in swinging corner couldn't have been placed better for the on rushing express train that is Martin Laursen.
With the full backing of the Holte End Villa looked in control.
Shorey and Young overlapped brilliantly from the full back positions to support the attack in their efforts to compensate for the lack of presence without Carew.
Ironically, It was an excellent defensive block from Luke Young that led to Ajax's 21st minute equaliser.
From the resulting corner, Thomas Vermaelen thumped a header in off the underside of Brad Friedel's crossbar.
If Young's delivery was good for Villa's opener, Urby Emanuelson's in-swinger was perfection, and his dead ball kicking proved a real hand full for the Villa back line all night.
Ajaz began to seize the initiative and could have scored in the 29th minute had their own players not got in each other's way when another Emanuelson corner evaded the Aston Villa defence.
Villa were quick to respond to the let off and set about restoring their lead. Ashley Young should have done better when he pulled a volley wide following good work down the right by Milner.
However, he made amends soon after having been released by a clever Shorey through ball. Young was once again too quick for Ajax full back Bruno Silva and his pull back fell to the left foot of Barry.
The midfielder was coolness personified as he stroked the ball first time beautifully past the hapless Vermeer to give Villa a 2-1 half time lead.
Merchant
It was enough to win the game and seemed to really deflate the Dutch giants as Villa dominated possession in the Ajax half for large parts of the second period.
O'Neill opted to push Barry further up the field to support Agbonlahor, who had been peripheral and isolated for large parts of the first half.
Agbonlahor was far busier thanks to the support and will feel he should have had a penalty in the 67 minute.
The pace merchant was tripped as he bore down on goal but his protestations were waived away by the Austrian referee.
As Villa tired, Ajax took the ascendancy. In the 78th minute Friedel could only palm a powerful long range shot from palm a powerful long range shot from Emanuelson back into his penalty area but the impressive Shorey was on hand to cover the rebound and hack the ball to safety.
Thanks to some excellent positional play by Cuellar and tireless closing down all over the pitch, Villa held on to complete a memorable victory.
O'Neill has an excellent track record against Ajax both as a manager and as a player and will revel in his decision to play his full backs as auxiliary wingers to supplement an attack lacking its focal point.
The only down side to the evening was Agbonlahor's exit on a stretcher in injury time. Though, it seemed to have only been a kick and nothing more serious.
The Villa boss was quick as always to praise his side and the importance of the victory: "We were terrific and fought back magnificently to get ourselves back in front after their equaliser." And also added: "They are a good side and pass very well indeed but we have excellent players and that should not be forgotten."
This was a big test for Villa and they came through it with flying colours. O' Neill had targeted seven points as being enough to successfully negotiate the group stages before the game and with three of those safely in the bag, the Claret and Blue army can look to the remaining games with real optimism.