Mitoma was forced to miss the Seagulls' 1-1 draw with Everton in the Premier League last weekend, but his problem was unrelated to the nasty challenge from Sheffield United's Mason Holgate a week earlier.
De Zerbi has since confirmed that the 26-year-old is nursing a back injury which is set to rule him out for at least a couple of months.
Speaking at a press conference ahead of Brighton's FA Cup fifth-round tie with Wolverhampton Wanderers on Wednesday, De Zerbi said: "A bad, bad situation. I think we've lost Mitoma until the end of the season.
"He has a back injury, it is an important problem. I think he will be out for two or three months and two or three months finishes the season."
Mitoma previously missed three Premier League games between late December and early January due to an ankle injury, and the Japan international subsequently emerged as a doubt for the Asian Cup.
However, Mitoma was fit to feature in Japan's last-16 and quarter-final fixtures before returning to Brighton earlier this month.
The winger is now dealing with a fresh injury setback at a time when Brighton are still competing in three different competitions - the Premier League, FA Cup and Europa League.
Brighton still have 12 games remaining in the Premier League and currently sit seventh in the table, five points behind Manchester United in sixth and eight points adrift of Tottenham Hotspur in fourth.
The Seagulls are also gearing up for a challenging Europa League last-16 tie against Roma, with the first leg in Italy taking place on March 7.
Mitoma has been a standout performer for Brighton since the arrival of De Zerbi, but he has only contributed with three goals and six assists in 26 games during a disrupted 2023-24 campaign.
In the absence of Mitoma, De Zerbi could turn to Barcelona loanee Ansu Fati, Simon Adingra or Julio Enciso to play on the left flank, the latter of whom has recently recovered from a long-term knee injury.
Another option for the Seagulls is versatile left-sided teenager Valentin Barco, who joined the club from Boca Juniors in January and is soon hoping to break into De Zerbi's first-team plans.
Meanwhile, De Zerbi has confirmed that attacking duo Evan Ferguson (ankle) and Joao Pedro (hamstring) will miss Brighton's FA Cup trip to Molineux on Wednesday, while Danny Welbeck and Joel Veltman require late fitness checks.
Solly March (knee), Jack Hinshelwood (foot) and James Milner (thigh) all remain in the treatment room, while Billy Gilmour will miss the match against Wolves as he is serving a three-game suspension following his red card against Everton.