Nick Ansell scored the only goal of the game as the Victory upstaged a youthful Atletico outfit, with coach Diego Simeone deciding not to field a host of stars, including Fernando Torres and Diego Godin, having faced Tottenham Hotspur two days earlier in the ICC.
The Victory looked strong early and almost took the lead through Fahid Ben Khalfallah, but his volley from an Oliver Bozanic corner sailed just over the crossbar.
But Atletico responded and Amath Diedhiou had a similar chance minutes later, before Javier Manquillo, Tiago and Rafael Borre forced Victory goalkeeper Lawrence Thomas into saves.
Kevin Muscat's side then lost winger Mitch Austin to an injury, having failed to recover from his opening-minute clash with Emiliano Velazquez, before Simeone replaced Tiago with Thomas Partey at the 35-minute mark.
And it was the Victory - slightly against the run of play - who eventually took the lead right on the stroke of half-time, with Ansell rising highest for a Ben Khalfallah set-piece to score with an emphatic header, beating Atletico goalkeeper Jan Oblak.
The hosts produced an impressive performance during the second-half against their more faniced opponents, while Thomas was kept busy in goals as the Victory maintained their lead.
Both coaches decided to make wholesale changes to their line-ups on the hour-mark - with nine in total across the two teams.
Despite the introductions of Saul and Diogo Jota, who was making his Atletico debut, the Spanish visitors were unable to trouble the Victory defence, with the Australian side holding out for on for another memorable win.
The victory is another boost for Kevin Muscat's men ahead of the A-League season and their FFA Cup Round of 32 clash with Newcastle Jets at Broadmeadow's Magic Park on Wednesday night.
Additional reporting: Omnisport