The popular duo, who were ranked 16th, lost 6-4 4-6 6-3 to unseeded Germans Yannick Hanfmann and Dominik Koepfer on Saturday.
Wildcards in last year's draw, Hijikata and Kubler secured a memorable first grand slam title when they accounted for Hugo Nys and Jan Zielinski in the 2023 final.
Their loss puts a dent in hopes of a local pairing capturing the men's doubles title for a third straight year, with Kubler and Hijikata having followed the equally unlikely charge by Nick Kyrgios and Thanasi Kokkinakis in 2022.
The Germans sent down 12 aces to five from the Australians and blasted 43 winners to 23 to wrap up the win in one hour and 51 minutes.
After levelling the match at a set apiece, Kubler dropped his serve in the third set for the fan favourites to trail 1-3, with Hanfmann, the world No.52 in the singles, consolidating.
That break was all the Germans needed with the veteran Hanfmann sealing the result with an unplayable serve down the 'T' to Kubler.
Fellow Australian pairing Max Purcell and Jordan Thompson also bombed out, falling 6-3 6-7 (5-7) 6-1 to Zielinski and Nys.
Matthew Ebden, half of the second-seeded pairing with 43-year-old Indian Rohan Bopanna, is the last Australian remaining in the men's doubles.
Ebden and Bopanna ended John Millman's career on Friday with a straight-sets win over the former US Open quarter-finalist and rising youngster Edward Winter.
In the mixed doubles on Saturday, wildcards Olivia Gadecki and Marc Polmans shocked fourth seeds Hao-Ching Chan from Taiwan and Mexico's Santiago Gonzalez.
The Australian duo won a thriller 6-4 6-7 (5-7) 12-10.
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