The Scotland international was replaced at half-time during the 3-1 loss at Manchester United with a back injury.
Goals from Robin Van Persie, Tom Cleverley and Wayne Rooney had United 3-0 up inside a hour before Frazier Campbell grabbed a consolation against his former club.
O'Neill said: "Steven hurt his back in the warm-up before the game. He twisted it in some way and it was causing him a few problems.
"It got more painful in the last 10 minutes of the first half and he couldn't continue."
O'Neill does not know how long Fletcher will be out of action, although clearly any injuries are a major blow to a club only a point above the drop zone and with an already weak squad.
"We would have to be seriously looking to try to strengthen our side," added the Irishman. "You only have to have look at the back of the programme to see that we don't have great numerical strength.
"But you are going to get injuries and suspensions. It is up to us to cope with them.
"It was going to be hard enough without giving a couple of poor goals away.
"But there is still a decent spirit about us. We lack many things in the game but the spirit is still there and has not been broken."