In 1501, two papal bulls were issued concerning the child, Giovanni Borgia. In the first, he was recognized as Cesare's child from an affair before his marriage. The second, contradictory, bull recognized him as the son of Pope Alexander VI. Lucrezia's name is not mentioned in either, and rumors that she was his mother have never been proven. The second bull was kept secret for many years, and Giovanni was assumed to be Cesare's son. This is supported by the fact that in 1502 he became Duke of Camerino, one of Cesare's recent conquests, hence the natural inheritance of the Duke of Romagna's oldest son. Giovanni went to stay with Lucrezia in Ferrara after Alexander's death, where he was accepted as her half-brother.

After the death of Lucrezia's second husband, her father, Pope Alexander VI, arranged a third marriage. She then married Alfonso I d'Este, Duke of Ferrara, in early 1502 in Ferrara. She had eight children during this marriage and was considered a respectable and accomplished Renaissance Duchess, effectively rising above her previous reputation and surviving the fall of the Borgias following her father's death.

Neither partner was faithful: beginning in 1503, Lucrezia enjoyed a long relationship with her brother-in-law, Francesco II Gonzaga, Marquess of Mantua. Francesco's wife was the cultured intellectual Isabella d'Este, the sister of Alfonso, to whom Lucrezia had made overtures of friendship to no avail. The affair between Francesco and Lucrezia was passionate, more sexual than sentimental as can be attested in the fevered love letters the pair wrote one another. The affair ended when Francesco contracted syphilis and had to end sexual relations with Lucrezia. This last assertion is problematic as Francesco had contracted syphilis before 1500 as it was known that he passed the disease onto his eldest son Federico Gonzaga who was born in 1500. Francesco did not meet Lucrezia until 1502.

Lucrezia met the famed French soldier, the Chevalier Bayard while the latter was co-commanding the French allied garrison of Ferrara in 1510. According to his biographer, the Chevalier became a great admirer of Lucrezia's, considering her a "pearl on this Earth".

After a long history of complicated pregnancies and miscarriages, on 14 June 1519 Lucrezia gave birth to her tenth child, named Isabella Maria in honour of Alfonso's sister Isabella d'Este. The child was sickly and – fearing she would die unbaptised – Alfonso ordered her to be baptised straightaway with Eleonora della Mirandola and Count Alexandro Serafino as godparents.

Lucrezia also had a love affair with the poet Pietro Bembo during her third marriage. Their love letters were deemed "The prettiest love letters in the world" by the Romantic poet Lord Byron when he saw them in the Ambrosian Library of Milan on 15 October 1816. On the same occasion Byron claimed to have stolen a lock of Lucrezia's hair – "the prettiest and fairest imaginable" – that was also held there on display.

One painting, Portrait of a Youth by Dosso Dossi at the National Gallery of Victoria, was identified as a portrait of Lucrezia in November 2008. This painting may be the only surviving formal portrait of Lucrezia Borgia; however, doubts have been cast on that attribution. Several other paintings, such as Veneto's fanciful portrait, have also been said to depict her, but none have been accepted by scholars at present.