Benfica finished third behind Celtic in Group G after the Scottish side beat Spartak Moscow 2-1 to end with 10 points, three behind Barca and two clear of the Portuguese team who will go into the Europa League
The La Liga leaders fielded a side full of youth-team players but all the home fans wanted to see was World Player of the Year Messi try to score the one goal he needs to match Gerd Mueller's record of 85 goals in a calendar year.
The Nou Camp's chants for Messi to join the game were answered in the 58th minute after Benfica had squandered a host of chances to take the lead.
The Argentine had a couple of efforts and broke through one-on-one with visiting keeper Artur in the 85th minute.
The goalkeeper collided with Messi as he tried to round him before catching the forward's attempted lob.
Messi dropped to the ground clutching his left knee and he was taken off on a stretcher with his hand covering his face, clearly in pain. The injury was later diagnosed as bruising.
"It's a blow [to the knee]. The doctors are looking at it and we'll know more after they have done the tests," Barca coach Tito Vilanova told Spanish television.
"It doesn't seem to be serious but we'll have to wait and see."
Vilanova rested regulars such as Victor Valdes, Xavi, Andres Iniesta and Cesc Fabregas in favour of a number of Academy players but it was a surprise that Messi was on the bench.
"You have to give these youngsters a chance. We want to see how they react at the Nou Camp, in a Champions League game," Vilanova added.
"It would be better to do it with them surrounded by more experienced players but we were already qualified.
"It wasn't a comfortable game for us."
Benfica forward Rodrigo fired wide of the post in a three-on-one chance with Barca reserve keeper Jose Manuel Pinto after only 12 minutes, as the hosts lacked their usual fluidity.
Pinto rose to the occasion, however, and tipped Lima's shot on to a post, and then pushed another from Ola John into the side-netting.
The chances came and went for Benfica but they started to fade in the second half until Messi had to leave the pitch.
Having played all their substitutes Barca were on the back foot with 10 men, and Benfica piled forward in search of a winner that would put them into the knockout round.
Maxi Pereira had their best chance but blazed a shot over the bar in the fifth minute of stoppage time and with it went their chances of qualifying for the next round.
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