The essay explores and examines in depth the connection between the ancient Grotto of Egeria on the Via Appia and the Grotto at the Medicean Villa Medici in Castello. This study deepens further this relationship, with new elements concerning the iconological level, and opens up a new horizon regarding the commission of the Medicean grotto and its patronage, focusing in particular on the role of Eleonora di Toledo in Cosimo I’s patronage. The link with the cultural Roman context of the 1530-40s is a fundamental point of view, according also to the contemporary Paduan cultural environment. This study proposes a new approach concerning the role of Benedetto Varchi in the initial history of the Medicean complex, strengthening the attribution to Varchi of the iconographic and iconological programme of the garden at Castello. The antiquarian value of the Grotto of Egeria as model for the Grotto at Castello reinforces the central position of the Medicean complex in the wider European garden history.