The 35-year-old fast bowler had been battling to be fit in time for Wednesday's crucial clash against India in Southampton after two defeats in two matches for South Africa.
But the seamer has been forced to admit defeat, leaving head coach Ottis Gibson's side short of firepower.
"Steyn has suffered a second shoulder injury which has not responded to treatment and has ruled him out of bowling for the foreseeable future," Cricket South Africa said in a statement.
South Africa have called up left-arm pacer Beuran Hendricks as a replacement, with Lungi Ngidi also nursing a hamstring problem.
But Du Plessis said despite their injury problems and their poor start to the tournament, South Africa were aiming to reach the last four of the round-robin event, in which all 10 teams play each other.
"Six games is the target, and there's seven games left," the skipper told reporters at the Ageas Bowl.
"It would be great if we could start that tomorrow. There's no easy games any more in the World Cup.... Whether it's winning or losing, the main thing is getting to the semi-finals."
Hashim Amla returns for the India game after missing the second match following a hit on the head during the tournament opener against England.
Du Plessis said it was important to stay "strong and positive" despite the challenge of working with limited resources.
"Naturally, guys will have a little bit of confidence that's been chucked away and that is normal, that is part of being human," he said.
Hendricks will arrive on Wednesday but will not be available to face India.
Du Plessis admitted South Africa's injury problems had forced him to draw up a new plan.
"I think we'll really have to look at that now, there's only 14 players to pick from now. There's not a lot of options for the bowling attack," he said.
"You have to decide whether you go with all your all-rounders in your team, bat deep and try to get a big score."