Wing Mirco Bergamasco and lock Marco Bortolami have been ruled out following injuries sustained in their 19-14 win over Tonga.
Bergamasco suffered a broken cheekbone and Bortolami sustained a dislocated clavicle in the bruising battle
The news is a significant blow to Italy's chances of qualifying for the quarter-finals, both players have been key figures for Italy, and were expected to start against Wales.
Now the quarter-final decider is a tougher challenge again for the Azzurri, who are also sweating on the fitness of wing Andrea Masi, who is working to shake off a hip injury. `
"We have a little bit of difficulty in naming our team. Hopefully, we'll get our team out in time otherwise we'll be subject to another fine (under International Rugby Board rules)," said Italy coach John Kirwan
Better news for Italy is that national captain Alessandro Troncon is expected to be fit for the match.