A princess from the House of Cybo-Malaspina, she was the daughter of Ricciarda Malaspina, Marquise of Massa and Lady of Carrara, and Lorenzo Cybo, Count of Ferentillo, who, from 1530 to 1541, also held, in a turbulent condominium, the sovereign titles of his wife.

Unhappily residing in a convent, Eleonora urged her uncle, Cardinal Innocenzo Cybo, to hurry the marriage process.

Eleonora, who was still childless, sought refuge in a convent and, having proved unable to recover her dowry, she eventually moved back to Tuscany settling with her father at Agnano, not far from Pisa.

Unhappy with the prospect of having to return to cloistered life, Eleonora sought the help of Grand Duke Cosimo I of Tuscany, a distant relative of her father's, who arranged her marriage to condottiero Chiappino Vitelli.

Eleonora's intellectual circle included notables such as poet Bernardo Cappello, humanist Ludovico Domenichi, and poetess Laura Battiferra, who dedicated sonnets to her.