Kyle Abbott, given a chance after South Africa rested its frontline pace attack, removed the Australian openers in the first and third overs of the innings on his way to 4-40. Spinner Tabraiz Shamsi took 3-36 as Australia stumbled after choosing to bat first at St. George's Park.
Australia was 49-5 before half-centuries by Mitchell Marsh (50) and Matthew Wade (52) saved the tourists from an even more modest total.
South Africa won with nearly 15 overs to spare, with captain Faf du Plessis leading the chase with 69.
Top-ranked Australia faces a series whitewash in the final game in Cape Town Wednesday.
Brief Scores
Australia: 167 all out in 36.4 overs (Matthew Wade 52, Kyle Abbott 4-40, Tabraiz Shamsi 3-36) lost to South Africa: 168 for 4 in 35.3 overs (Faf du Plessis 69, Chris Tremain 2-48) by six wickets.