Former world number one Hewitt, who retired six weeks ago at the Australian Open, replaced Sam Groth as John Peers's doubles partner and dragged Australia back from two sets down with an inspired returning game on the temporary grass court at Kooyong Lawn Tennis Club.
But the Bryans silenced the home crowd by breaking Peers's serve in the second game of the final set and the veteran Americans then served brilliantly to deny the hosts another break-point.
Serving to stay in the match at 5-2 down, the Australians saved two match points on Hewitt's serve but conceded in the next game when Bob Bryan served to 40-0 and his brother smashed the winning volley.
Australia will pin their hopes on world number 20 Bernard Tomic upsetting 11th-ranked John Isner in the first of the reverse singles on Sunday.
The pair have played only once before, with Isner a comfortable winner at Delray Beach in 2012.
Groth, who was well beaten by Isner in the opening singles on Friday, was named to play Jack Sock in Sunday's final singles rubber during the draw on Thursday.
But Australia can still change their nomination, meaning 35-year-old Hewitt could return to the court against Sock, who lost his first singles tie in four sets to Tomic.
(Reporting by Ian Ransom, editing by Nick Mulvenney)
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