The Blaugrana star was brought on in the second half as a substitute but was forced to withdraw late in the game, leaving his chances of equalling Gerd Muller's record in doubt
Coach Tito Vilanova chose not to start the Argentine attacker in their final group match but introduced him 13 minutes into the second half, replacing Rafinha, as the Blaugrana pushed for a win.
But with five minutes left - and the game still goalless - the Ballon d'Or favourite went down clutching his leg and had to be taken off the Camp Nou pitch by medics after colliding with Eagles goalkeeper Artur.
Following his withdrawal, neither team were able to break the deadlock, and Benfica's inability to score condemned them to the Europa League while Celtic advanced to the round of 16 after they beat Spartak Moscow 2-1.
Many suspected Wednesday night would be the moment when Messi equalled - or even surpassed - Gerd Muller's record of 85 goals in a calendar year.
However, should Messi's injury rule him out for even a fortnight, the winter break that Spanish clubs partake in could allow the legendary German striker's record remain intact.
Barcelona fielded a largely second-string XI against the Portuguese outfit as first place in Group G was already assured, so their stalemate is unlikely to trouble Vilanova.
A serious injury to his star player, on the other hand, would no doubt trouble the 44-year-old, despite their six-point lead at the top of La Liga.