Brignone defeated Olympic giant slalom champion, Swede Sara Hector, by 0.57 seconds.
Alice Robinson of New Zealand finished nine-tenths of a second behind the winner in third place.
“It wasn't easy in the second run, but the slope was incredible,” said Brignone, who earned his 14th career victory in giant slalom.
“I really wanted it, I’m so happy. It was a tough race, but I felt really good, I wanted to fight and I wasn't too stressed.”
No Italian woman had won the race near the Austrian capital of Vienna since Karen Putzer's triumph in 2002. The Semmering race takes place every two years.
At 34 years and 5 months, Brignone improved her record as the oldest Women's World Cup winner.
Brignone mastered a tricky passage towards the end of a course where Italian coach Giorgio Pavoni put the gates in the first round.
“It was good, rhythmic and with a lot of turns,” said Brignone, Olympic vice-champion in 2022 and world champion in the discipline in 2011.
Defending champion Lara Gut-Behrami, second after the first run, was slowed in the second when she snagged a gate with her left arm midway through. The Swiss fell to ninth position.
Brignone has won four of the last five giant slaloms.
She reached the top of the discipline rankings this season, overtaking Hector.
Canada was also represented by Cassidy Gray (20th) and Britt Richardson (23rd).
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