The aim of the study was to characterize the heterotrophic microbiota in surface samples and in the air of the Roman catacombs of St. Callistus and St. Domitilla. The microbiotas inhabiting different environments and substrates, including plaster, marble and tufa in illuminated and dark sites, were studied. Microbial groups examined were aerobic microorganisms, anaerobic and facultatively anaerobic bacteria, proteolytic microorganisms, actinobacteria, yeasts and moulds. Filamentous actinobacteria, Streptomyces spp., were dominant in the biofilm samples. 16S rDNA sequence similarity analysis indicated that many of the isolates were novel species. Gram-negative bacteria were a minority among the isolated bacteria: few slime forming bacteria or types most closely related to them, like Pseudomonas spp., Stenotrophomonas nitritireducens, Sinorhizobium morelense and Bosea thiooxidans, were isolated. Fungi, mainly white Lecanicillium psalliotae, Torrubiella spp., Beauveria alba and L. aranearum, were isolated from both air and biofilm samples.