The pop queen had been due to start at 9pm local time, but thousands of fans at the Entertainment Centre were told that the Material Girl would not start before 10.30pm at the earliest.
Still no Madonna on stage in Brisbane. One new Mum with a babysitter waiting just gave up. #RebelHeartTour— Jen Dudley-Nicholson (@jendudley) March 16, 2016
"Madonna was 2 and a half hours late, queen of unpunctuality thank you for blessing me with lateness xoxo," another added.
Fans have commented via social media that the venue supplied refunds for the event.
"It was just announced that #Madonna is not going to arrive till 10.30 at the EARLIEST (1.5hrs late). Hate it. #RebelHeartTour," Jess Ism said in a series of tweets.
"Can't even get a cup of tea coz the line is so long. I'm feeling the 'Mad' in #Madonna No British star would pull this shit #RebelHeartTour," Ism added.
"It's just inconsiderate. It's mid week, we have kids and work to consider. If you print 8pm start then don't come in at 11:30pm," Tanya O'Dea said on Facebook.
Madonna finally made her first appearance at 11.22pm, more than two hours after the original set time.
Her tardiness comes almost a week after the star left annoyed fans waiting four hours in the Melbourne rain before arriving on stage for her intimate Tears of a Clown show at around 12.50am last Friday morning.
The 57-year-old, who last set foot in Australia 23 years ago, thanked the near-capacity Brisbane crowd for their patience on Wednesday night but laughed off her late arrival without offering an apology.
"I'm never late, well hardly ever late," she said."You were here too early. You should have taken your time doing your hair and makeup, then I wouldn't have been late."
"No one wants to come early."
One fan said on Facebook she was disappointed to hear from a staff member that Madonna was at the venue, but simply refused to go out onstage.
While it seemingly took only seconds for the crowd to forgive the performer and get into the show, many were angered to find public transport to the Entertainment Centre had stopped shortly after midnight - an hour before the concert eventually ended.
"@Madonna By 2am no trains no buses very few taxis or uber. Without your own car you would be stranded!" Alison Hale said.
And that was not the only complaint. Others claimed the wait wasn't even worth it.
"Leaving now from the concert. Worst concert ever @Madonna Pitch issues, lip syncing,boring foul language. Every one is walking out!" a disappointed fan said.
"It's true that Madonna's concerts have finished late. They should have finished in 1998," one fan tweeted.
But not all fans were disappointed by the pop sensation, with true Madonna fans rushing to her defence.
"It has taken 23 years to come back to Australia who cares if she's late .. A true fan wouldn't so why are you even commenting?" Jules Brashaw said.
"What are you complaining about ? You got a refund ! Eat ya birthday cake and s*** up ! You are not a fan at all ! @Madonna is Queen!" another said.
Madonna is set to perform a second show in Brisbane on Thursday night before ending the tour in Sydney on the weekend.