The visitors were leading Australia 23-14 with 15 minutes remaining at Perth's Subiaco Oval when winger Bryan Habana, playing in his 100th test, was shown a yellow card for a high tackle on Wallabies winger Adam Ashley-Cooper.
Although Australia only managed a penalty while Habana was off the field, the last-ditch defending with 14 men took its toll and, two minutes from the end, the Wallabies found space to send Rob Horne in for a converted try to snatch a 24-23 win.
South Africa now head to New Zealand, where a similarly contentious call for a tackle on Dan Carter by Bismarck Du Plessis last year ultimately cost the Springboks the chance of upsetting the All Blacks in Auckland.
With number eight Duane Vermeulen also penalised for a high challenge in Saturday's match, coach Heyneke Meyer was asked whether he thought the Springboks were being targeted for their tackling by officials.
"I'd love to answer that question but I'm not going to put my foot it in," he told reporters in Perth.
"At the end of the day, we respect all the referees, he has to make choices and we're never going to use it as an excuse. Again with Duane, I thought it was an excellent tackle but that's the way the referee sees it.
"I'll keep my integrity intact, we take it on the chin. On the day, I think we really played well but didn't get the result."
There was little doubt, though, that Meyer thought the decision had cost his side the game, disrupting his plans to bring loose forward Warren Whiteley off the bench for his first cap and sapping the defensive effort.
"Unfortunately, you can't play with 14 men in the last 10 minutes," he said.
"I thought the guys were brave, we just needed one more second, but we couldn't keep them out.
"I thought we controlled the second half very well, playing against the wind."
The loss means South Africa's hopes of winning a first southern hemisphere title since 2009 now all but certainly rest on getting a result against the All Blacks next week.
"It's a defeat and it really hurts," Meyer said. "We now need to set our sights on our next match, which is our third away from home on the trot, and rectify matters against New Zealand in Wellington.
"It will take a monumental effort and we've got a lot of respect for New Zealand, but I believe we can beat them there."
(Reporting by Nick Mulvenney, editing by Patrick Johnston)
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