Meristematic black fungi are a highly damaging group of microorganisms responsible for the deterioration of outdoor exposed monuments. Their resilience to various stresses poses significant challenges for removal efforts. This study focuses on the community of meristematic fungi inhabiting the external white marble of the Cathedral of Santa Maria del Fiore, where they contribute to its darkening. Twenty-four strains were isolated from two differently exposed sites of the Cathedral, and their characterization was conducted. Phylogenetic analysis using ITS and LSU rDNA regions revealed a wide diversity of rock-inhabiting fungal strains within the sampled areas. Eight strains, belonging to different genera, were also tested for thermal preferences, salt tolerance, and acid production to investigate their tolerance to environmental stressors and their interaction with stone. All tested strains were able to grow in the range of 5–30 °C, in the presence 5% NaCl, and seven out of eight strains were positive for the production of acid. Their sensitivities to essential oils of thyme and oregano and to the commercial biocide Biotin T were also tested. The essential oils were found to be the most effective against black fungi growth, indicating the possibility of developing a treatment with a low environmental impact.