It was the first loss for Team USA in a major international tournament or exhibition since the 2006 world championships against Greece.
The Americans had since gone undefeated for over a decade - a span that includes three Olympics and two World Cups.
It was also Australia's first-ever win against the United States in 31 all-time meetings.
San Antonio Spurs guard Patty Mills scored a game-high 30 points on 10-of-22 shooting - including the Aussies' last 10 points - to lead the Boomers past the world's top-ranked nation.
"It's a building block for us," Mills said postgame. "We're taking it step-by-step and understand that this is a process."
"Some of it is expected with a new group that's trying to learn about each other and learn a system,'' Team USA head coach Gregg Popovich said, according to The Associated Press. "So it's not surprising. But the Aussies gave us a great lesson as far as where we want to be and how you have to play in this kind of a competition.''
The United States have one final exhibition game against Canada on Monday, while the Aussies await the latter in their World Cup opener Sept. 1.