Cosimo I contracted a morganatic marriage with Camilla Martelli on 29 March 1570 on the advice of Pope Pius V, and this allowed him to legitimize their daughter on the principle of per subsequens.

Cosimo I's older children resented their father's second marriage, and after the death of the Grand Duke in 1574, they imprisoned Camilla in the Florentine convent of Murate.

After this, it was decided to arrange her marriage with a member of the House of Este with the purpose to improve the relations between both families and break the isolation of the Grand Duchy of Tuscany from the other Italian states.

To celebrate this event was represented the comedy "l’Amico Fido", written by Giovanni de' Bardi and with the lyrics of Alessandro Striggio and Cristofano Malvezzi,[7] and in Ferrara the poet Torquato Tasso dedicated a Cantata to the newlyweds.

On 15 January 1598 the Duchy of Ferrara was officially abolished and returned to the Papal States, despite Cesare's attempts to obtain the help of the major European powers.

[12] Nevertheless, she coped with her motherly duties with her numerous offspring and showed herself as a clever and far-sighted ruler when in January 1601, in the absence of her husband (who was in Reggio) the heavily pregnant Duchess took the position of Regent.

However, Virginia was unable to control her unpredictable anger fits: when in March 1608 her confessor, the Jesuit Jerome Bondinari, claimed that she was possessed by the devil, the Duchess attacked him violently with shouts and nearly beat him to death with a stick.