She was the illegitimate daughter of Francesco d'Este and Maria Folch de Cardona.
She and her sister Bradamante (born 1559) were legitimised by both pope Gregory XIII and Alfonso II d'Este.
She was also left a palace that year by her father, who began building it in 1559; it was called after her Palazzina Marfisa d'Este and was slowly abandoned after her death.
On 30 January 1580, she married Alderano Cybo-Malaspina, heir apparent of the Principality of Massa and Carrara.
After the Duchy of Ferrara's devolution to the Papal States in 1598, due to the absence of legitimate male heirs of the House of Este, Marfisa refused to join her family in Modena, and remained in Ferrara with her husband in the palace she inherited from her father.