Club captain Puyol had arthroscopic surgery on the cartilage in his right knee in March and Mascherano damaged ligaments in the same joint in Barca's 2-2 quarter-final first leg draw at Paris Saint-Germain on April 2.
"Puyol won't be available for the game in Munich and we don't know yet about the return leg [on May 1], it will depend on how he recovers and the doctor's decision," Roura, standing in for coach Tito Vilanova while he recovers from cancer treatment, told a news conference.
"Mascherano is coming along well but we still don't know whether he can play in the return leg," he added.
Barca, chasing a third European crown in five years, have several options to choose from to partner Gerard Pique in central defence, including full-back Adriano, youth-team product Marc Bartra and midfielders Alex Song and Sergio Busquets.
Eric Abidal, who returned to action this month after a liver transplant, is another possibility although he may need more time before he is back to full fitness.
"We have to take it step by step," Roura said of the Frenchman.
"It's normal that he is keen to play, just like his team-mates.
"He has worked extremely hard and he is more ambitious all the time."
One player who appears certain to have recovered from injury before Tuesday is Barca's Argentina forward Lionel Messi.
The World Player of the Year rejoined the rest of the squad in training on Friday as he continues his recuperation from a hamstring strain sustained in the game at PSG.
"Messi is in good shape," Roura said. "He is back working with the group again and he has coped well with his recovery and is doing all he can to be fit."
