She was the fourth of eight children and the second daughter born to Cosimo II de' Medici, Grand Duke of Tuscany and his wife Maria Magdalena of Austria.

The wedding celebrations were accompanied by much spectacle and pageantry, including the performance of Marco da Gagliano's opera La Flora, composed especially for the occasion.

According to portraiture and the sources of the time, Margherita was a beautiful woman, as well as amiable and well-educated.

Unlike his predecessors, the duke was very devoted and loyal to her and there were never any sources that claimed Odoardo had illegitimate children.

She served in co-regency with her former brother-in-law, her son‘s uncle, Francesco Maria Farnese.