Within the huge hypogean complex of San Callisto, Rome, Italy, the cubicles of the Roman Catacombs of Santi Marco, Marcelliano e Damaso (4th century AD) contain decorated surfaces, such as the painting depicting the Twelve Apostles over the arcosolium and the peacocks in the niches within the namesake cubicle. Biological deterioration due to growth of bacterial, fungal and phototrophic organisms has relevant implications for preventive conservation strategies in hypogeum systems. Taking into account the dual perspective ‘heritage conservation and promotion, this work presents the results of the biological investigations carried out at the entrance of these Roman Catacombs and close to a skylight from which the sunlight partially illuminates the corridor. A microclimate monitoring was complementarily performed, based on the evidence that the indoor microclimate is not always necessarily constant and microbial colonies frequently change their appearance throughout the year, thereby suggesting a dependence on the local environmental parameters. In-situ observations and laboratory characterization of the biodeteriogens revealed the dominance of Basidiomycetes at the entrance, and a compact biofilm mainly consisting of filamentous cyanobacteria and other microorganisms in the lighted areas of the catacombs. Taking into account the specific microclimate, monitored in terms of relative humidity and temperature, preventive solutions were suggested. The results obtained from testing this monitoring approach encourage further implementation in similar hypogean contexts.