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The preservation of cultural heritage, including ancient photographic materials, is of paramount importance in the field of conservation science.In this context, the microbial diversity of ‘Skull and Crossbones’, a 1983 photograph by Robert Mapplethorpe printed on silver gelatine, was assessed. We employed both culture-dependent and culture-independent methods to characterize microbial communities inhabiting this artwork. Vibrational Raman micro spectroscopy and FT-IR spectroscopy were utilized to assess the chemical degradation condition and characterize the chemical components of the silver gelatin print. The combination of molecular sequencing methods (Sanger and HTS approach) and non-invasive vibrational spectroscopy yielded valuable insights into the microbial communities thriving on photographic material and the chemical degradation of the print. Isolated fungal strains were added to the Fungal Collection at the University of Naples Federico II, and their deteriorative potential was investigated by adding substrates, commonly used in canvas photographs to the culture media. These results establish a link between microbial communities colonizing ancient photographic materials, paper decomposition, and the enzymatic patterns of the retrieved microorganisms. This information is invaluable for understanding and addressing biodeterioration progression on valuable works of art, such as historical photographs, which remain understudied.