Villa claimed another huge European scalp as they stormed into the quarter-finals of the NextGen Series.
Michael Drennan and Brad Lewis were on target as Tony McAndrew's team came from behind to record a memorable victory over last season's tournament runners-up Ajax.
Drennan's first-half strike took his NextGen goal tally to six, making him the competition's current top scorer.
This incredible result at the Dutch side's famed De Toekomst headquarters ensured the claret and blues will advance to the last eight for the second year running.
Villa made five changes from their last NextGen Series outing - the 5-1 win at Sporting Lisbon back in November which set up this last 16 fixture.
Brad Watkins replaced Craig Hill in goal, while Lewis, Derrick Williams, Lewis Kinsella, and Jordan Lyden came in for Janoi Donacien, Liam Bateman, Malcolm Melvin and Jordan Graham.
The only survivors in the Ajax line-up from the last meeting between the two clubs - a 3-0 Villa victory at Villa Park last season - were goalkeeper Micky van der Hart and skipper Danzell Gravenberch.
Although the away side made a solid start, it was the hosts who took the lead against the run of play when Djavan Anderson produced a cool finish from just inside the box on 14 minutes.
The visitors looked to stage a quick response and Drennan was unlucky to see a shot go just past the post.
However, on 35 minutes Drennan equalised with his 13th goal in all competitions this term.
The Republic of Ireland U19 striker showed his clinical edge with a powerful effort that found its way past Van der Hart.
After the break, it was Villa who continued to look the more dangerous and on 64 minutes they took the lead.
Lewis was found unmarked in the Ajax box by Samir Carruthers and the defender chipped the ball into the far corner of the net.
Ajax pushed hard for an equaliser in the closing stages but Villa held on to deservedly seal their place in the quarter-finals.
AJAX: Van der Hart, Tete, Gravenberch, Riedewald, Rosheuvel, Boccara, Lazic, Anderson, Bitter, El Hasnaoui, De Sa. Subs: Alblas, Van den Boomen, Noordhoff, Keskin, Kuipers, De Bondt, Becker.
VILLA: Watkins, Webb, Kinsella, Lyden, Lewis, Williams, Barton, Carruthers, Drennan, Burke, Grealish. Subs: Bateman, Toner, Calder, Crowley, Abdo, Crooks.