The 20-time winners of the competition had steadied the ship somewhat in recent months following a disastrous start to the 2023-24 season, but their campaign plumbed new depths in the humble surroundings of Stadion Galgenwaard in Utrecht.
John van't Schip's side initially looked to have spared their blushes by recovering from two goals down with a quickfire double in the final seven minutes of normal time.
However, Mats Grotenberg popped up with a 93rd-minute winner for the home side to hand USV Hercules their biggest ever victory, and arguably the biggest shock in the history of the competition.
No amateur team had even scored against Ajax in the KNVB Beker since 2017 prior to this evening's game, but that record was ended after only 16 minutes when Tim Pieters gave the hosts a shock lead.
Even accounting for their historically poor form in the opening months of the campaign, there was an expectation that last year's beaten cup finalists would fight back, having gone into the match with six wins and two draws from their last eight domestic outings - an unbeaten run which stretched back to their heavy defeat at the hands of rivals PSV Eindhoven in October.
It was USV Hercules that flexed their muscles once again, though, as Pieters doubled both his personal tally and his side's lead shortly after the hour mark.
Ajax were still two goals down heading into the final seven minutes of normal time, but Brian Bobbey gave Dutch football's most successful club hope by halving the deficit in the 83rd minute.
Chuba Akpom then restored parity in the 89th minute to seemingly break Hercules hearts, only for there to be one dramatic final twist in the epic tale.
Grotenberg wrote his name into Hercules history forever with his 93rd-minute winner, handing the fourth-tier side a famous victory over the biggest name in Dutch football.
Indeed, excluding penalty shootouts it was the club's first victory in the cup against any team since March 1937, while Ajax fell to defeat in the competition at the hands of amateur opposition for the first time ever, from 30 previous such matches.
Ajax have now lost 14 matches in 2023, only ever losing more in a single calendar year once - 17 in 1964.