Six months after her brother Philip, Margaret married John of Castile and Aragon on April 3, 1497. He, however, died on October 4 of the same year. A few weeks after, Margaret gave birth to a stillborn child.
After her return to the Netherlands, she became the godmother of her nephew, the subsequent Charles V. In 1501, Margaret married Philibert of Savoy, who died only three years after.
Her father could not convince her to marry for a third time. In 1507, Margaret was declared governess of Habsburg Netherlands and tried to regain her heritage, the Dukedom of Burgundy. She died in Mechelen on December 1, 1530.