The world number 11's win on a steamy afternoon at Melbourne's Kooyong Lawn Tennis Club sealed the tie 3-1 with a singles rubber in hand, while silencing a raucous crowd of yellow-clad fans.
"Our team prepared very, very well and I think it showed out here," Isner, who closed out the contest with his 49th ace on match point, said courtside.
The win will take some heat off U.S. captain Jim Courier, who came into the tie on the back of two first-round exits from the team tournament.
"We knew it was going to be very difficult," said Courier, whose team will face Croatia or Belgium in the last eight in July.
"Our team certainly came good, John stepped up so big ... and finally cracked it there in the breaker which was really impressive."
The United States, Davis Cup champions 32 times, arrived Down Under early to prepare and had first practice on Kooyong's temporary grass court even before Australia's team had assembled in Melbourne.
The Lleyton Hewitt-captained Australia lost young gun Nick Kyrgios to a virus in a major blow in the leadup but Isner led off superbly for the Americans with a dominant win over Sam Groth in the opening singles.
He also appeared set to cruise to another emphatic win on Sunday before wobbling when serving late in the fourth set.
Tomic finally broke the towering American on the fifth set point but neither player offered a look at the other's serve in the decider.
Isner's chief weapon would ultimately prevail, as he blasted a string of huge serves to seal the tiebreak and condemn Australia to a first round exit after their run to the semi-finals last year.
(Editing by ....)
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