The 32-year-old has been replaced in coach Cesare Prandelli's selection by Juve team-mate Angelo Ogbonna, who returns to the Azzurri set-up for the first time since a friendly victory over San Marino in May.
An Italian Football Federation statement read: "Andrea Barzagli has left the national side's retreat. The Juventus defender is struggling with a recurrence of tendon problem and will not recover for the games against Bulgaria and the Czech Republic."
Italy tackle Bulgaria in Palermo on Friday before facing the Czech Republic at Turin's Juventus Stadium on September 10 in a World Cup qualifying double-header. Two wins could be enough to see Prandelli's men win Group B and book their place at next year's finals in Brazil.