Willian was introduced in the second-half of Brazil's 2-0 win over Argentina on Tuesday with the Brazil football federation (CBF) confirming on Wednesday he pulled his hamstring during the match.
While not a serious injury, Willian's recovery time means he has been ruled out of the final as the hosts look to claim the Copa America for the first time since 2007.
The 30-year-old had been used solely as a substitute at the tournament to date but did score during his side's 5-0 win over Peru in the group stages and converted a penalty in the quarter-finals against Paraguay.
A Brazil football federation spokesman told the AFP that Willian felt "pain in his hamstring" and was sent for tests on Wednesday, where it was confirmed he had pulled a muscle.
Willian was himself a late call up to Brazil's Copa America squad with the attacker replacing Neymar after the PSG star was ruled out of the tournament through an ankle injury.
Despite being without Neymar, Brazil have still managed to seal their place in the final and are yet to concede a goal at the tournament.
They will now face Peru in the final on Sunday after the Incas saw off Chile 3-0 on Wednesday with Peru last appearing in a Copa America final back in 1975.
Brazil will however back themselves on home soil and will be buoyed by their 5-0 demolition of Ricardo Gareca's side in the group stages.
While Willian's Copa America campaign has come to an end, he's set for an interesting return to club land with Chelsea close to confirming Frank Lampard as their new manager.
The Brazilian attacker is clearly a fan of Lampard's and believes he looms as an ideal replacement for Maurizio Sarri.
“He is a legend,” Willian told football.london.
“He was an extraordinary player for the club, winning every possible title.
“He deserves so much respect and backing for what he has already achieved at the club. If Chelsea do bring him in, he will be very welcome."