Referee Guido Winkmann, who was also on his way to the tunnel before the VAR intervened, ordered the players to return so that Mainz's Pablo De Blasis could take and score a penalty. After looking at replays of a cross from Mainz's Daniel Brosinski striking Freiburg defender Marc Oliver Kempf on the hand, Winkmann awarded the spot-kick and the players were "hauled back to the pitch".
Players from both sides had already been on their way back to their respective dressing rooms when the decision was made.
Mainz midfielder Pablo de Blasis scored the penalty to put the hosts 1-0 up.
Then the second half was delayed because fans hurled an avalanche of toilet rolls onto the pitch, in protest of the Monday night kick-off.
Mainz began the day three points behind their relegation rivals and a win would see them move into 16th place and shove their rivals into a relegation playoff place.
Mainz remained the stronger team in the second half, and De Blasis wrapped up victory 11 minutes from time after an embarrassing error from Alexander Schwolow.
Speaking to Eurosport, Freiburg sporting director Jochen Saier said the side would have to "accept it with a heavy heart": "It leaves a bitter taste".
The VAR system, which has been used in the Bundesliga all season, has caused numerous controversies during testing in the FA Cup in England this season.The Premier League recently voted against introducing it for the 2018/19 season.
A Bundesliga match descended into chaos as the referee awarded a penalty during half-time after checking the VAR.