The Southern Stars fell 16 runs short of the 147 needed at Chelmsford with the night belonging to England star Sarah Taylor who scored an impressive 77 off 57 balls.
It was the highest score by an Englishwoman in the shortest form of the game.
England lead the mixed-format Ashes eight points to four with two points up for grabs in each of the remaining two Twenty20 games.
Australian captain Jodie Fields acknowledged the hosts made to a flying start on Tuesday and it was hard to come back after they posted 4-146.
"It would have been nice to restrict them to a few less but we did think we'd make that total if we batted smartly," Fields told Sky Sports after Australia finished at 5-131.
"But tonight, it was England's day."
The sole Test of the mixed-format Ashes was drawn in mid-August before Australia won the first one-day international and England won the next two ODIs.
Momentum is clearly with the hosts but Fields remains confident the Aussies can turn things around in the final two Twenty20 clashes on Thursday and Saturday.
"We don't give up in these situations," she said.
"We only need two wins to retain it. We are not that far off - we just have to improve in small areas and, if we do that, I'm sure we can come out fighting in Southampton."
England captain Charlotte Edwards insisted Taylor's 77 was the difference between the two teams on Tuesday.
Taylor, 24, was bowled by Erin Osborne on the last ball of the English innings attempting to hit her seventh boundary of the night.
A run-out by Rachael Haynes earlier contributed to Australia's four wickets.
The Southern Stars made a terrible start to the run chase when Meg Lanning was run out for just two by an Arran Brindle direct hit.
Fellow opener Alyssa Healy was out shortly afterwards for six and, although Cameron tried to revive Australia's fortunes, she fell for 35 and it was looking grim.
Ellyse Perry smashed 30 not out off 28 balls but it was too little too late.