Marta converted a penalty kick in the 72nd minute and the Pride beat the Spirit 3-2.
The third-place Pride (11-8-6) are undefeated in their last four visits to Audi Field dating to 2022.
The loss had no impact on the standings for the Spirit (12-5-8), who already locked up the second seed with one regular-season match remaining.
The Spirit took a 1-0 lead when Kerry Abello scored an own goal in the 35th minute.
Abello quickly made amends and tied it 1-1 in the 38th, with a shot on her left foot from just inside the box.
Sofia Cantore put the Spirit back ahead in the 42nd. The Italian international flicked the ball into the goal from a tight angle at the near post with her heel.
At halftime, Pride head coach Seb Hines brought on Marta for Carson Pickett.
Orlando tied it just after halftime when Narumi Miura scored an own goal in the 46th minute via a shot from Marta, who had only been on the field for 52 seconds.
Kysha Sylla fouled Ally Watt in the box, and Marta stepped up to convert the penalty kick.