The AC Milan midfielder was taken off after just eight minutes following a challenge from Alex Pearce and the team doctor confirmed he has suffered a broken leg
The AC Milan captain went down after challenging for the ball with Alex Pearce and required several minutes of treatment on the pitch before being replaced by Alberto Aquilani.
Italy team doctor Enrico Castellaci confirmed after the match that Montolivo has suffered a broken leg and will be unable to travel to the finals next month.
"Montolivo has a fracture on his tibia, he probably will need an operation. He can't go to Brazil with us," he said.
The 29-year-old was involved from the start in something of an experimental line-up from coach Cesare Prandelli, but took a hit to his ankle after just eight minutes at Craven Cottage following Pearce's challenge.
Italy players looking on appeared concerned over the midfielder's injury and medical staff were quick to attend to him on the pitch before ultimately carrying him off on a stretcher.
Montolivo made seven appearances for the Azzurri during their World Cup qualifying campaign and was likely to be an important figure for Prandelli as one of the more experienced players in the camp.
Italy - who drew 0-0 on the night with Ireland - face England in their World Cup opener in just two weeks.