Culley fell to the turf in the second quarter at the MCG on Friday night and was helped to his feet before limping off the ground.
The 23-year-old was soon ruled out and cut an emotional figure when comforted by teammates at the three-quarter time huddle.
Culley previously ruptured the anterior cruciate ligament in his right knee in 2023 while playing with West Coast.
He will have scans on Saturday to determine the extent of the latest damage.
"Obviously it doesn't look great when a player goes down like that and can't return," Demons coach Steven King said after his side's 19.12 (126) to 11.6 (72) victory.
"Unfortunately, we're fearing the worst but he'll get scans tomorrow and I'll cross all my fingers and toes that it's a good result.
"We feel for Jai but hopefully it's better than anticipated."
Melbourne medical staff appeared to be assessing Culley's left knee at quarter-time, but King backed their judgement.
"I'm not sure there, it didn't come up to me, but no, we back our staff in completely," King said.
"It's unfortunate. He's worked really hard over summer and he's a really important part of what we do, being on a wing with his size and athleticism.
"Hopefully he's okay, but we've got a great medical team and the welfare of our players is more important than the result of the game for us.
"We take care of our people and they would've made the right call."
Culley joined Melbourne as a pre-season supplemental selection last year and has established himself in the team, playing all seven matches this season.
He was awarded a contract extension earlier this month, securing his playing future until the end of 2028.
"He worked as hard as anyone to secure the spot and the contract he did," King said.
"I'm sure whatever the outcome he'll attack it and come back better."
14°C