The biological colonization of rocks in the Cave of Bats (Cueva de Los Murciélagos, Zuheros, Spain) was studied in order to reveal the diversity of microorganisms involved in the biofilm formation. The culturable, metabolically active fraction of biodeteriogens present on surfaces was investigated focusing on morphological, ultrastructural, and genetic features, and their presence related to the peculiar environmental conditions of the underground site. PCR-ITS analysis and 16S rDNA sequences were used to clusterize and characterize the isolated strains. The presence of bacterial taxa associated to the photosynthetic microflora and fungi within the biofilm contributed to clarify the relationships inside the microbial community and to explain the alteration observed at the different sites. These results will contribute to the application of more successful strategies for the preventive conservation of subterranean archaeological sites.