The main objective of this work was the comparative analysis of a large number of bacterial strains isolated from biodeteriorated surfaces in three different sites, namely the catacombs of St. Callistus in Rome, Italy, the catacombs dedicated to St. Agatha in Rabat, Malta and the Cave of Bats in Zuheros, Spain. Our results showed that even considering only culturable chemoorganotrophic bacteria the variability is very high, reflecting the great variety of microhabitats present. Hence any strategies to prevent, control or eliminate the biofilm-embedded microbiota from an archeological surface should take into account a number of considerations as stipulated in our study.