The Argentinian World Player of the Year collapsed to the ground clutching his left knee after colliding with Benfica goalkeeper Artur and looked to be in severe discomfort as he was taken from the pitch.
Barca said on their Twitter feed he had suffered bruising on the outside of the knee and would undergo further tests to determine the extent of the damage.
The game ended 0-0 with Barca already through to the last 16 as group winners and Benfica forced to settle for a place in the Europa League following Celtic's late 2-1 victory against Spartak Moscow.
Messi remains one goal short of Gerd Muller's record of 85 goals in a calendar year set in 1972.
Elsewhere, an own goal saw Juventus go through to the knockout stages as Group E winners as they edged out Shakhtar Donetsk in Ukraine.
Olexandr Kucher inadvertently turned Stephan Lichtsteiner's cross into his own net in the 56th minute, seeing the Italians leapfrog Shakhtar at the top of the group.
Both teams struck the post but Juventus could just about claim to be worthy winners as they had the better of the chances and ensured most of the game was played in Shakhtar territory.
Bayern Munich sealed top spot in Group F as they coasted to a 4-1 victory over BATE Borisov at the Allianz Arena.
Substitute David Alaba completed the rout with seven minutes remaining before Egor Filipenko grabbed a BATE consolation.
By then, both sides had been reduced to 10 men with Bayern's Jerome Boateng seeing red for a poor challenge on Artem Kontsevoy in the 51st minute and Denis Polyakov following 18 minutes later for a second bookable offence.
The victory saw Bayern pip Valencia to top spot, despite the Spaniards winning 1-0 in Lille thanks to a 37th-minute penalty from Oliveira Jonas after Marko Basa had impeded him.