The Serie A club said Saturday that Pjaca injured his left knee in training the previous day and tests revealed he has torn the anterior cruciate ligament.
Pjaca, who is on loan from Juventus, will see a surgeon in the next few days to plan the necessary treatment.
Fiorentina coach Stefano Pioli said: “He is a very unlucky guy. What happened yesterday to him affected everyone. Even today the guys were still a bit shaken. We are very close to him. He is a strong lad and he will overcome this umpteenth awful injury.”
Pjaca was sidelined for almost 10 months after tearing the ACL in his other knee on international duty in March 2017.
The 23-year-old Pjaca joined Fiorentina on loan from Juventus for this season with the option to make the deal permanent.
Pjaca has made 24 appearances for Croatia and was part of the squad which reached the final of the 2018 World Cup.
The team posted a message of support to Pjaca on its official twitter account.