Black patinas are a very common deterioration phenomenon of lapideous artworks in outdoor environments. These substrates, exposed to sunlight, atmospheric and environmental agents (i.e. wind and temperature changes), represent an ‘extreme environment’ and can be colonized by microorganisms characterized by a huge versatility and adaptability. It is known that black patinas are made of a wide variety of microorganisms, but the morphological plasticity of most of these microorganisms not always allows the identification by optical microscope observation. This study performed Next-Generation Sequencing (including shotgun and amplicon sequencing) in order to characterize the black patina of the travertine muraglioni (embankments) of Tiber River in Rome (Italy). Overall, the sequencing confirmed the relevance of coccoid and filamentous Cyanobacteria observed by optical microscope, but also carried out the rich diversity of bacterial and fungal communities of black patina and showed a more convenient and informative landscape of the total microbial community. The knowledge of the taxa presents in the patina and of their ecological requirements could be necessary for the choice of appropriate conservation treatments.