The 33-year-old former wunderkind was bidding for his first main draw berth in five years, needing three wins in qualifying to earn a start.
But Tomic was unable to back up his first-round victory, falling to Great Britain's Arthur Fery in their Wednesday clash 6-4 6-2.
It was 23-year-old France-born Fery's second successive win over an Australian after accounting for Edward Winter in round one.
While Tomic spoke of his joy to be playing in front of home fans after his first round straight-sets win over Bolivian 25th seed Hugo Dellien, he declined any interviews: "Not today", he said as he brushed past media.
Ranked 184 and playing most of the year on the ATP Challenger Tour, Tomic is coming off his most successful season in seven years.
The former world No.17 competed in 35 tournaments in 2025 amassing 44 wins, 35 losses, making almost $360,000.
Still, it could be the last time Tomic, who last played in the Open in 2021 when he reached the second round, is seen at Melbourne Park.
In other results, Olivia Gadecki also was in no mood for talking after letting a golden opportunity slip against former world No.3 Sloane Stephens.
The 23-year-old Queenslander had an early break in the deciding third set before she allowed 2017 US Open champion Stephens back into the match for a 3-6 6-3 6-3 victory.
The only major winner playing in qualifying this week and a 2013 Australian Open semi-finalist, 32-year-old Stephens was sidelined for seven months last year with a foot injury.
Arriving at Melbourne Park on the back of a 13-match losing streak, her WTA ranking dropped outside the top 1000, forcing Stephens into qualifying for the first time since Wimbledon in 2012.
Storm Hunter, Dane Sweeny and newly-engaged pair Maddison Inglis and Jason Kubler at least gave home fans something to cheer, all marching into the final round of qualifying with impressive wins.
On the comeback trail after a year out nursing a ruptured Achilles tendon, Hunter downed Serbian 27th seed Lola Radivojevic 6-2 7-6 (7-1), while Sweeny advanced with a 6-4 6-4 victory over Swiss Jerome Kym.
Inglis outlasted American Claire Liu 7-6 (8-6) 2-6 6-4, while her fiance Kubler took out Lithuanian Vilius Gaubas 7-5 6-2.
Hoping to play in his second Australian Open, the 24-year-old Sweeny was happy with his game.
"Definitely a step up from my first round, I'm playing with a lot more courage and conviction," he said.
"First round I was a little bit hesitant and that was the main goal today, win or lose was to play on my terms and I think I did a really good job.
"I played very tactically smart and he had the firepower, but I think I just kind of swindled in with his skills."
Another young Sydneysider James McCabe suffered a tough super tiebreak loss to rising Norwegian star Nicolai Budkov Kjaer 4-6 6-3 7-6 (10-6).
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