In reply to South Africa's 242, Australia were cruising at 158 for none, but then lost all 10 wickets for just 86 runs to lead by only two on the first innings.
Steyn left the field for scans on his troublesome right shoulder before lunch, just after claiming the wicket of Australian opener David Warner for 97, and did not return for the remainder of the innings.
In his absence, Vernon Philander (4-56), Kagiso Rabada (2-78) and debutant spinner Keshav Maharaj (3-56) exposed the home side's batting frailty.
Warner appeared certain to score his fourth Test century at the WACA Ground, but was caught at first slip by Hashim Amla and his dismissal sparked a remarkable period of play.
Just eight runs later, Steyn left the field mid-over, after appearing to reinjure his troublesome right shoulder in an around-the-wicket delivery to Usman Khawaja.
One run later, Khawaja joined Warner back in the pavilion when he was clean-bowled by young paceman Rabada for four.
South African hopes were further boosted when Australian captain Steve Smith was the victim of a highly contentious lbw decision for a duck.
He was visibly shocked to be adjudged out by veteran umpire Aleem Dar, who has already made a number of controversial decisions in the match.
The Australian skipper called for a decision review, which showed the ball just clipping the outside of the stumps, which is out under new rules introduced recently.
Steyn hurt his shoulder with the fourth ball of his 13th over, having been by far the most menacing of the South African bowlers to that stage.
The 33-year-old missed several months of cricket earlier this year with the injury and immediately left the WACA to have scans.
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