Urbanization and urban regeneration can significantly impact cultural heritage, but a greater knowledge of the past natural and historical features is needed to value the past and understand the present. The lost Villa Peretti Montalto in Rome, once located in the area that corresponds to the current front side of Termini station, deserves great attention due to its cultural value. This work aimed to provide a floristic and functional reconstruction of the gardens of the villa during the XVI and XVII Centuries. From several bibliographic and iconographic sources, a critical analysis and interpretation of plant names was conducted. A list of 87 species and their location in the different garden sectors, during different periods with their specific uses, is provided. The arboreal design was made by classical species in the Roman context, like Cupressus sempervirens, Pinus pinea, Quercus ilex, and Ulmus glabra. In addition, ancient lost varieties of fruit trees (mainly Pyrus communis and Ficus carica) and several species of conservation interest were found. The knowledge of the ancient flora in historical gardens could be a key tool in urban greenery planning and touristic and cultural valorization.