Tito Vilanova chose not to start with the Argentine forward in the team's final group stage game, but brought him on midway through the second half as the Blaugrana pushed for a win.
With five minutes left - and the game still goalless - the Ballon d'Or favorite went down clutching his leg and had to be taken off the pitch at Camp Nou by medics, not long after he wasted an opportunity to give the Liga giants a late lead.
"Messi has a bruise on the outside of his left knee. He'll undergo further tests to determine the exact extent of the injury," was the message from Barcelona's official Twitter account following the game.
After the star substitute's withdrawal, neither side was able to break the deadlock and the clash finished 0-0, eliminating Benfica into the Europa League while allowing Celtic to advance after it beat Spartak Moscow.
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 a couple of weeks, the winter break that Spanish clubs partake in could allow the legendary German striker's record remain intact.
Barcelona fielded a largely second string squad against the Portuguese side as first place in Group G was already assured, so its stalemate with Jorge Jesus' side is unlikely to trouble Vilanova.
A serious injury to his star player, on the other hand, would no doubt trouble the head coach of the Catalan giants, despite their huge lead at the top of La Liga this season.