The Brazil international went down in the 30th minute of the league fixture at Bernabeu and was struggling to hold back the tears when he was stretchered off the field.
There was immediate concern surrounding a potential ACL issue for Militao, and the worst has now been confirmed.
Real Madrid's statement also revealed that the 26-year-old has damaged his meniscus, and he will be out for the remainder of the 2024-25 campaign.
Los Blancos, who beat Osasuna 4-0 on Saturday afternoon, have not placed a timeframe on a potential return for the defender, but it is likely that he will be on the sidelines for at least the next nine months due to the damage.
Militao suffers ACL injury in Real Madrid's clash with Osasuna
"After the tests carried out today on our player Eder Militao by the Real Madrid Medical Services, he has been diagnosed with a complete rupture of the anterior cruciate ligament with involvement of both menisci in the right leg. Militao will be operated on in the coming days," read a statement from the European and Spanish champions.
Militao was out for the majority of last season with an ACL injury, and he will now have to go through the entire process again before hopefully returning to action during the 2025-26 season.
"The demands of this calendar do not allow the players to rest just enough, they arrive with less rest to the matches. It's a general problem, not just ours," Real Madrid head coach Carlo Ancelotti told reporters after the clash with Osasuna, with the capital team returning to winning ways after successive losses to Barcelona and AC Milan.
Will Real Madrid sign a centre-back in January?
Real Madrid remain without the services of David Alaba, with the Austria international missing since last December with an ACL injury.
Los Blancos did not replace Nacho over the summer, meanwhile, so the club were already short in the middle of the defence, and there is now major pressure on Antonio Ruidiger to stay fit.
Aurelien Tchouameni is also an option in the middle of the defence, while Raul Asencio came off the bench to make his debut for the club following Militao's injury blow.
When asked about potential arrivals in January after Saturday's game, Ancelotti said: "In January we will see. I don't think about signings because we have to prepare games with the ones we have... today Raul Asencio gave a good contribution."
Real Madrid also lost Rodrygo and Lucas Vazquez to muscular injuries in their four-goal win, and it is believed that the pair will be on the sidelines for the next four weeks.