Botanical gardens in city centres are green islands, offering a sanctuary for diverse plant and fungal species amidst urban landscapes. Although lichens are not usually a primary focus in botanical gardens, these spaces can serve as important refuges. In this work, the epiphytic lichen biota has been investigated in the Botanic Garden of Pisa, to uncover the hidden elements of biodiversity of botanical gardens in terms of non-target organisms. A total of 57 epiphytic lichen taxa and one non-lichenized fungus were recorded, including several threatened species. Two species, Arthopyrenia platypyrenia and Coenogonium tavaresianum, are new records for Tuscany, while Lecania cyrtellina is so far known in the region as occurring only in the Botanic Garden of Pisa. Lichen colonization reflects the richness of tree species, while the diffused presence of nitrogen-tolerant lichens could be associated with urban environmental conditions. Comments on Arthopyrenia platypyrenia, Coenogonium tavaresianum, Gyalecta liguriensis, Lecania cyrtellina, Lecanographa amylacea, Micarea misella, Porina borreri, and Waynea stoechadiana are given. Keywords: Biodiversity, Botanic Garden of Pisa, Epiphytic lichens, Species conservation, Tuscany.