Sitting in the relegation playoff spot at kick-off, Mainz stunned the visitors by racing into a three-goal lead inside just 23 minutes.
Leandro Barreiro opened the scoring on 12 minutes, before Lee Jae-sung grabbed two goals in three minutes as the hosts tore into a much-changed Dortmund side.
Mainz, who are now eight games unbeaten since losing 8-1 to Bayern Munich in March, realistically need just a point from their final game of the season at Wolfsburg to secure another season in the German top flight.
With Dortmund's Champions League qualification for next season now guaranteed, and a Champions League final against Real Madrid on the horizon, Dortmund coach Edin Terzic rang the changes.
Defender Nico Schlotterbeck was the only starter from the team that won in Paris on Tuesday and though Jadon Sancho, Ian Maatsen and Julian Brandt came off the bench at half-time, they could not turn the tide.
Dortmund now sit four points behind fourth-place RB Leipzig, who drew 1-1 with Werder Bremen earlier on Saturday.
But one spot behind Dortmund in sixth, Eintracht Frankfurt are a distance adrift of Terzic's side, and drew 1-1 at Borussia Moenchengladbach in their penultimate match of the season.
At the other end of the table, Mainz's victory leaves Union Berlin, who squandered a 2-0 lead at Cologne earlier in the day to lose 3-2, languishing in the thick of the relegation fight.
Cologne Downs Union
Cologne teenager Damion Downs came off the bench to score a 92nd-minute winner, keeping their hopes of a miraculous great escape alive.
Union had raced into a 2-0 lead inside 19 minutes with Robin Knoche's thumping header and Kevin Volland's penalty putting the Berliners in complete control.
A Florian Kainz penalty shortly before half-time gave Cologne hope but Union's lead remained intact until the 87th minute, when Cologne staged a stunning turnaround.
Steffen Tigges scored from close range to make it 2-2 and, with Union still reeling, 19-year-old substitute Downs converted Linton Maina's cross to raise the roof and give his side an invaluable win.
"We're overjoyed," Cologne captain Kainz told Sky Sport Germany.
"It's nerve-wracking to play like that, when you absolutely have to win the game. To turn it around, it was a hugely important victory. Unbelievable."
But Mainz's victory means the best Cologne can hope for on the final day is to secure a relegation play-off place at Union's expense.
Union, who started the season in the Champions League but are now on a miserable run of two points from their last seven games, face a tricky season finale against Freiburg -- who remained in the last European spot after drawing 1-1 against Heidenheim on Saturday.
Cologne travel to Heidenheim on the last day needing to muster another victory and hope that Freiburg win in Berlin.
Goal difference and goals scored would also come into play in this scenario, with Cologne needing a four-goal swing to finish above Union.
One of Germany's 'yo-yo clubs', Cologne are looking to avoid a record seventh relegation since their first in 1998.
Bochum, who host champions Bayer Leverkusen on Sunday and travel to Werder Bremen on the final day, need a point from one of those games to ensure their Bundesliga survival.
