Maria was the eldest and only surviving child of Francesco IV Gonzaga, Duke of Mantua and Montferrat and Margaret of Savoy (1589–1655), daughter of Charles Emmanuel I, Duke of Savoy and Infanta Catherine Michelle of Spain. Maria was already Duchess of Montferrat since 1612. This led to the War of Mantuan Succession.
Maria and Charles had two children:
Charles Gonzaga (1629–1665) married Archduchess Isabella Clara of Austria and had issue;
Eleanor Gonzaga (1630–1686), who married in 1651 Ferdinand III, Holy Roman Emperor and had issue.
She died in 1660, aged 51.