Biofilms colonizing ancient limestone Mayan monuments in Uxmal (Yucatán, Mexico) were characterized for their microbial composition and differences using phospholipid fatty acid (PLFA) analysis and single-strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR)-amplified genes of the rRNA small subunit. Biofilms 1–7, displaying different macroscopic characteristics, were drawn from interior and exterior limestone walls, which were characterized by different microenvironmental conditions. Inner surfaces that were exposed to low light intensity and higher water availability supported a higher biofilm biomass, probably indicating that these environmental conditions are more suitable for biofilm growth and development. Sequencing of SSCP profiles from the biofilms showed that bacteria affiliated with the phyla Proteobacteria, Bacteroidetes and Cyanobacteria colonized both internal and external surfaces, although certain Nevskia and Salinibacter were particularly associated with internal biofilms. In contrast, a particular biofilm community composed almost exclusively of Rubrobacter-related bacteria was found in only one exterior partially shaded sample (biofilm 3), characterized by high illumination and low water content. No specific organisms were detected in biofilm 7. The PLFA profiles suggested that cyanobacteria were the biomass-dominant group in most biofilms, except for biofilm 3 (exterior) where actinobacterial markers were detected in significant proportions. Interestingly, most of the detected sequences were related to halophylic bacteria, although the similarity of these clones to known sequences in databases was low (< 94%). This finding suggests that much bacterial novelty, probably of halophylic nature, remains to be identified in these biofilms. Our study suggests that water availability and light regime appear to be the main environmental determinants defining boundaries for biofilm formation, while substratum salinity appears to be an important abiotic factor that influences biofilm community structure in these specialized microbial habitats.