Aims: To determine the major components of total and metabolically active microbial communities of yellow and grey colonizations threatening the conservation of palaeolithic paintings in Altamira Cave (Spain). Methods and Results: Micro-organisms present in yellow and grey colonizations were determined from DNA analysis with those showing metabolic activity determined from RNA analysis. Microbial community fingerprints were obtained by denaturing gradient gel electrophoresis (DGGE) and 16S rDNA libraries were constructed from PCR amplified products. Proteobacteria was the most frequent bacterial phylum. Other phyla detected from RNA-based microbial surveys were Acidobacteria, Actinobacteria, Firmicutes, Nitrospirae and Gemmatimonadetes. The detected metabolically active micro-organisms represented only a fraction of the total bacterial community present in the studied colonizations as compared from DGGE analysis. Conclusions: The major bacterial participants in the development of yellow and grey colonizations in Altamira Cave were determined using RNA-based molecular techniques. Micro-organisms showing undetectable activity represent a potential risk for the conservation of these paintings if environmental conditions experience variations. Significance and Impact of the Study: Caves with palaeolithic paintings are affected by microbial deterioration. Assessing the composition of the microbial communities colonizing these caves represents a first stage to understand and control these colonizations.