The Frenchman has been fighting his way back from a calf injury and looked on course to return to the squad ahead of Saturday’s clash with Real Madrid.
Mathieu has been absent for the last five La Liga games as the Bluagrana continue to struggle for defensive options.
The club have confirmed he will not be available for game at the Nou Camp after suffering a recurrence of the injury, which is likely to keep him out for another 20 days.
A Barcelona statement said: "During the final stages of his recovery, Jeremy Mathieu has suffered a recurrence of the injury to the soleus in his right leg.
"According to the FC Barcelona Medical Services, this means he'll be sidelined for a further 15 to 20 days.
"That means the French defender will be unavailable for the forthcoming games against Real Madrid, Borussia Monchengladbach and Osasuna."