The French-Croatian combination, who linked up for the first time at Melbourne Park, comfortably ensured a local team lost the final for the second year running with their 6-3, 6-4 victory on Friday.
Neither Fourlis or Kubler had won a Grand Slam mixed doubles match before they joined forces and were handed a wildcard for this year's Open.
The pair saved match points in three of their first four matches and took a "nothing to lose" attitude into the final against the fifth seeds.
But they were broken early in the first set and were ultimately outclassed by their more experienced and aggressive opponents.
Both Fourlis (shoulder) and Kubler (knee) have overcome career-threatening injuries in recent years.
"We started on Court 15 in the sticks and we slowly made our way up to Rod Laver Arena, even playing a match at 1am, so we've been through everything and it's been so much fun," Fourlis said.
Kubler, who played alongside Mladenovic in junior ranks, said: "It's a shame we couldn't get the win.
"It would've been a pretty cool story for us but, far out, we had so much fun."
Mladenovic is no stranger to breaking Australian hearts on the big stage, having upset a tearful Ash Barty in the 2019 Fed Cup final in Perth.
Friday's victory was Mladenovic's third major mixed doubles title after she partnered Canadian Daniel Nestor to win Wimbledon in 2013 and the Australian Open the following year.
The 28-year-old has also won five women's doubles Grand Slam titles, saluting at Melbourne Park in 2018 and 2020.
Dodig has won the French Open (2018 and 2019) and Wimbledon (2019) mixed doubles crowns with Latisha Chan.
He has also won two men's doubles Grand Slam titles, including last year's Australian Open.
Dodig and Mladenovic exchanged plenty of messages before finally deciding to link up for this year's tournament.
"What a way to finish our first time on court together," Mladenovic said.
"It was special for us playing against an Australian team."
The last Australian combination to win the mixed doubles crown at Melbourne Park was the pairing of Matt Ebden and Jarmila Gajdosova in 2013.