Background: Urban surface stones in Mexico City are exposed to a temperate climate and a range of atmospheric conditions ranging from mildly impacted to heavily polluted areas. In this study, we focused on the characterization of the cultivable fungal component of selected biological patinas in the surrounding area of Chapultepec castle, a historic monument in Mexico City. Thirty four representative fungal isolates selected based on distinctive differential macroscopic characteristics out of a total of 300 fungi, were characterized using morphological and molecular approaches. Results: This identification strategy based on the combination of phenotypic- and molecular-based methodologies allowed us to discriminate the fungal community in some cases down to the species level. Conclusions: The characterization of this mycoflora revealed the presence of a complex fungal community mainly represented by filamentous fungi belonging to the genera Fusarium, Trichoderma, Aspergillus, Cladosporium, Alternaria, Mucor, Penicillium, Pestalotiopsis, and the dimorphic fungus Aureobasidium, along with the yeast Rhodotorula. A specific distribution of fungi could be observed based on the type of biological patina analyzed.