The Italian pairing of Stefania Constantini and Amos Mosaner, who are also the world champions, cruised to an 8-4 victory over Koreans Jeong Yeong-seok and Kim Seon-yeong in the morning session.
However, a poor start in their game against Canada's Brett Gallant and Jocelyn Peterman ended their record, after they had made it through the only two competitions they played together - the 2022 Olympics and the 2025 World Championships - undefeated.
An inch-perfect play from Peterman left the Italians trailing 5-0 after the first end and the home favourites quickly unravelled, with Mosaner missing a string of shots as they reached the halfway mark trailing 6-2.
That deficit proved too much for Italy, who eventually conceded at 7-2.
"It went terrible in the first end for me. We gave up five in the first end, but I have to accept I missed the shots and they played better. We have to accept the loss and we try to start better tomorrow," Mosaner said.
The Canadians, making their mixed doubles debut at the Games, produced an impressive display on Thursday as they also beat Norway's two-time Olympic medallists Kristin Skaslien and Magnus Nedregotten in the afternoon session.
Asked how it felt to end Italy's unbeaten record, Gallant said: "It feels great, obviously. Going into that game, you really only have one option, you have to play really well because they're such a strong team.
"Defending world champs, defending Olympic champs. You have to get off to a great start, play really well. I'm just proud that we were able to execute that plan tonight."
Canada's win moved them level on three wins from three matches with Britain's Jennifer Dodds and Bruce Mouat, who beat the Czech Republic and Estonia on Thursday.
Switzerland and the United States occupy the remaining two playoff spots.
In the evening session, Estonia's Marie Kaldvee and Harri Lill claimed their country's first-ever curling victory at the Olympics in any category, beating Sweden's brother-sister duo of Isabella and Rasmus Wrana 7-5.