Needing only to take the baton around the track in the mixed 4x100m relay, Australia blew their chance to top the night in the finale of the new concept at Melbourne's Lakeside Stadium.
Jack Hale and Fabrice Lapierre botched the handover from second to third leg and Australia were disqualified.
After some initial confusion, Australia was given a score of -10, rather than the 40 points teams receive for finishing sixth.
As a result, the Bolt All-Stars -- who won the race with Bolt running the third leg -- finished with 930 points to Australia's 895.
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But meet officials reviewed the decision and ruled that a disqualification was appropriate.
"I don't know what happened," the world record holder at 100m and 200m said. "I've got to talk to the organisers and figure it out, but I'm sure we won the night."
In a bid to sell the concept globally, Bolt is fronting the All-Stars -- including athletes from Jamaica, the US and Kenya -- against teams representing Australia, England, New Zealand, Japan and China in the inaugural three-night event.
"I don't like the fact that we're leaving it that late," the Jamaican sprinter said.
The 30-year-old eight-time Olympic gold medallist suggested he might compete in more than one event in the final of the series this Saturday.
"I think a few of the guys will have to double up on Saturday. I'll probably have to double up."
But Bolt said he was feeling good about his own form, despite never having run competitively before at this time of year.
"I felt good. I came down here to get a few runs in. I'm not in the best of shape -- I'm just trying to run myself into shape -- and the more I run the better I feel," he said.
China won three events on the night and finished third on the points table with 855, just ahead of England on 850.
New Zealand won two events and finished fifth overall with 845 points. The Bolt All-Stars also scored a narrow 30-point victory over Australia last Saturday, 1080 to 1050.
Nitro Athletics involves a mixture of traditional and modified events, including mixed gender relays and an elimination mile, designed to provide non-stop action over a two-hour period
The All-Stars sit on 12 points with Australia on 10 after two second placings. Should Australia win on Saturday and the All-Stars finish second, both teams will finish with a total of 22 points.
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