Considered one of the most beautiful women of her time, after the death of her first husband, she was regent of San Martino all’Argine, Isola Dovarese, Rivarolo and Commessaggio from 1605 to 1613, due to the fact that her eldest son was still a minor.

Her disastrous second marriage to a distant cousin resulted in an accusation of witchcraft and an Inquisition process initiated at the request of her second husband's relatives, including his brother, Ferdinando I, which culminated in her complete rehabilitation.

She played a fatal role in the extinction of the main branch of the House of Gonzaga, which initiated the beginning of the War of the Mantuan Succession.

Because her oldest son was still a minor, she became regent of his domains, which included the Lordships of San Martino all’Argine, Isola Dovarese, Rivarolo and Commessaggio.

In 1609, she also became the regent of the Principality of Bozzolo, the Lordship of Rivarolo di Fuori and the County of Pomponesco when her still underage son inherited these domains from his paternal uncle, Giulio Cesare Gonzaga.

In 1613, her oldest son, Scipione, finally attained his legal majority and took over the government of his domains, after which Isabella retired to San Martino, near Mantua.

The Duke turned to the Bishop of Mantua with a request to declare the marriage invalid, in order to return Vincenzo to the cardinalate; his position was shared by his wife Catherine de' Medici, who even tried to convince her mother Christina of Lorraine to obtain the support of the Grand Duchy of Tuscany in this matter.

At first Vincenzo desperately resisted, but when he was banished to Goito and deprived of a number of material advantages, he broke down, and in December 1616 agreed with his older brother's demands to have his marriage to Isabella annulled.

Ferdinando I, using the help of his wife's cousin, Queen Marie of France, received the conclusion of the Sorbonne professors about the possible annulment of the marriage because of the close relationship of the couple; however, this wasn't enough.

Ferdinando I, childless in his marriage, understood that his younger brother was his heir, and wanted Vincenzo to marry soon to secure the survival of the dynasty; so, after an unsuccessful attempt to poison Isabella, the Duke accused her of witchcraft.