"Sergio Busquets and Eric Abidal will not feature in tomorrow's match," Barca said in a statement.
"The midfielder has not recovered from the (groin) injury that kept him from playing this weekend, while the Frenchman has picked up a calf injury."
Busquets is a crucial part of the Barca side in the often unsung defensive midfield role and without him their chances of overturning a 4-0 deficit from last week's first leg in Munich are severely dented.
The Spain international missed Saturday's 2-2 La Liga draw at Athletic Bilbao, when his place was taken by Alex Song. The Cameroon international turned in a decent performance but in general has struggled to make his mark since joining from Arsenal at the end of last season.
Abidal, who has only recently returned to competitive action after a liver transplant, was unlikely to feature against Bayern.
Barca's hopes of making next month's final at Wembley and winning a third Champions League crown in five years appear remote after they were comprehensively outplayed by the German champions in the first leg.
Only three clubs have overturned a four-goal first-leg deficit in UEFA competition and current Bayern coach Jupp Heynckes was on the losing side on one of those occasions.
The 67-year-old was in charge of the Borussia Moenchengladbach side that beat Real Madrid 5-1 at home in the 1985-86 UEFA Cup third round only to lose the return game 4-0.
Elimination for Barca on Wednesday would leave La Liga as their only chance of silverware this season.
They have an 11-point lead over current champions Real Madrid with five games left and are close to a fourth title in five seasons.
However, a domestic league crown would be seen as a poor return for a team who have set a new standard for European clubs in recent seasons and won the Champions League in 2009 and 2011.
"We are Barca and we cannot give up on a tie, there are still 90 minutes left," coach Tito Vilanova said.
"I don't think Bayern believe it is over either though they are better off than we are," he added.
"If we lose, at least we have to do it with our heads held high and playing on until the end of the match, not throwing in the towel and making our fans proud of us."