It was Mohun Bagan's second straight loss as the side looked disjointed while ace forward Odafa Okolie struggled to find the target.
The visitors took the opportunity to strike in the 27th minute when Douhou Pierre gave the lead with a stunner.
Mohun Bagan tried their best to come back but the dogged Pune FC backline led by Chika Wali thwarted all their efforts, while James Moga doubled the lead in the 68th minute as they kept penetrating.
Mohun Bagan did make a return when Odafa struck in the added time (90+4th) but it was too late.
The win took Pune (46 points) to five points behind Churchill Brothers who require one point from two matches to clinch the title for the second time.
Mohun Bagan (19) remained at 12th place as they still need three points from three matches to avoid relegation.