Stand-in skipper Marsh himself seemed to have laid the foundations for victory with his blistering opening knock of 60, but after he had perished needlessly to the spinning wiles of Kehav Maharaj, none of his team could cement the partnerships to get them anywhere near the hosts' 298-run target.
After watching the world champs collapse to 230 all out off just 41.2 overs, Marsh sighed: "I'm pretty disappointed, South Africa outplayed us. It was a good game of cricket, but we fell well short.
"We had our opportunities, I am probably going to sound like a broken record speaking about having partnerships in our batting, but we weren't able to form many tonight."
The South Africans had set a challenging target after being inserted by Marsh, compiling 8-297 thanks largely to a brilliant 112 from Matthew Breetzke and a spectacular 75 off 59 balls from pace-bowling allrounder Marco Jansen.
Marsh, standing in as skipper again for the rested Pat Cummins, then led from the front in response with his 36-ball pyrotechnics, which featured three sixes, one of which sailed for 104 metres over the dressing room roof at Kingsmead and landed at the roadside.
He shared a blistering opening stand of 90 in just 9.2 overs with Travis Head, but like Head (36), Cooper Connolly, who'd looked supreme in making 38, and Matt Renshaw (44), he got out to an airy shot when the Australians were up with the required rate and under no real pressure.
The superb spinning of the two slow left-armers Maharaj (4-20) and Bjorn Fortuin (2-34) soon put them under that pressure, though.
Discomfited, Josh Inglis got out to a poorly executed reverse sweep the eighth ball he faced and Australia lost their last six wickets for just 54 to allow the Proteas to draw first blood in the three-match series.
The key moment may have been when Jansen took a sensational running, diving, one-handed catch in the outfield, stretching out his long telescopic arm to scoop up the ball just above the turf to get rid of key man Renshaw off Gerald Coetzee. It left Australia on 7-215, and there was no way back.
So, after the three-match sweep of the Zimbabwe series, this really did feel like a proper test of Australia with the defence of their 50-over World Cup in southern Africa just a year away.
The first part of the reconnaissance mission demonstrated just how much they still have to work on, and the weakness against spin was again shown up even while South Africa had five top-line pace bowlers sidelined through injury.
Man-of-the-match could easily have been Jansen, with his one wicket, four catches and big-hitting or the superb 36-year-old veteran tweaker Maharaj, but batting at No.4, Breetzke's superb 106-ball knock, before being run out by Connolly, deservedly won the day.
Allrounder Jack Edwards had an uncomfortable debut -- 0-56 off 10 overs, followed by scoring five off 15 balls -- but there was encouragement again in the display of paceman Nathan Ellis, who, fresh from his outstanding series against Zimbabwe, was again the bowling pick with 3-57.
Allrounder Cameron Green, back in the side after missing the series against Zimbabwe as he recovered from a shoulder injury, had a poor day with the bat, falling for five while trying to belt Maharaj over cow corner and getting caught in the deep.
The opening loss has really put pressure on Australia to turn the series round at The Wanderers in Johannesburg on Sunday, when Cummins is set to be recalled. They haven't won a bilateral series against South Africa since 2014.
