Jose Mourinho's 100th game as Roma coach ended in disappointment as his club lost their coveted Champions League spot on goals scored.
Just days ago, Roma were comfortably sitting in third place in Serie A but Juventus jumped ahead after their 15-point penalty was suspended.
On Sunday, Milan's win at Lecce further tightened the competition for the top four.
Atalanta took the lead with a well-executed volley from Mario Pasalic after 31 minutes, and Rafael Toloi added a second goal from a corner in the 74th minute.
Roma showed resilience in the second half, with Lorenzo Pellegrini scoring off the post in the 83rd minute to give them hope.
However, their hopes were short-lived as a handling error by Roma goalkeeper Rui Patricio gifted a goal to Dutch midfielder Teun Koopmeiners just seconds later.
With only seven games left to play, Atalanta remains in the top-four race, four points behind Milan and Roma.
The pressure is now on for Roma as they prepare to face Milan in a crucial match on Sunday, hoping to regain their spot in the coveted Champions League positions.