The 17-year-old was forced off in the latter stages of the league game in Madrid, and Barcelona head coach Hansi Flick admitted after the clash that there was huge concern surrounding the injury.
The extent of the damage has now been revealed, with Bernal set to undergo an operation later this week before a further update on his situation is provided.
However, due to the seriousness of the injury, it is highly unlikely that the midfielder will be able to return to the field until the 2025-26 campaign.
"Tests carried out on Wednesday have shown that the player Marc Bernal has a ruptured anterior cruciate ligament in his left knee and an associated injury to the lateral meniscus," read a statement from Barcelona's official website.
Bernal suffers ACL injury against Rayo Vallecano
"In the coming days he will undergo a surgical procedure and when completed an update will be provided.
"Bernal started the game against Rayo Vallecano in week of La Liga and in injury time of the second half he was involved in a passage of play that led to him ending the game injured.
"A setback for the young midfielder who had started all three league games for the blaugranes so far his season."
Bernal has started all three of Barcelona's La Liga games this term, with the Spaniard benefitting from the club's injury problems in midfield, but he will now be working to return for the 2025-26 season.
Will Bernal's injury lead to Barcelona signing a new midfielder?
Gavi and Frenkie de Jong are still on the sidelines through injury, while Ilkay Gundogan has completed a return to Manchester City.
As a result, Pablo Torre, Marc Casado, Pedri and Fermin Lopez are currently Barcelona's only fit central midfielders, leading to suggestions that the club could sign a new player in that area before the end of the transfer window.
However, Flick said after the 2-1 victory over Rayo Vallecano that his side 'are covered' in the middle.
"This is very close to the match. Now we have to think about everything, but at the end of the day we have many players," he told reporters. "We have one match more before the international break and then hopefully players coming back. We can cover the No.6 [position] no problem."
Bernal is one of seven Barcelona players currently on the sidelines, with Andreas Christensen, Eric Garcia, Gavi, De Jong, Ronald Araujo and Ansu Fati also unavailable.