The knowledge about the microbial communities present in mural paintings is of utmost importance to develop effective conservation and mitigation strategies. The present paper describes a methodological approach for the detailed characterisation of microorganisms thriving in mural paintings by combining culture-dependent methods that allow the identification of microorganisms capable of growing in the laboratory conditions and to obtain high cell densities for further studies, and culture independent methods, such as denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The coupled use of culture-dependent methods and DGGE does not give enough information to investigate the diversity and abundance of microorganisms present in wall paintings. Pyrosequencing, a novel molecular technique, used here for the first time in this area of research, allowed the identification of a large number of microorganisms, confirming some already identified by the cultivation-dependent methods such as fungi of the genera Penicillium and Cladosporium, but also providing a great contribution in the identification of several genera and species, not previously identified in these artworks, giving also a detailed overview of contaminants which was not possible with the other approaches. The results obtained on several mural painting samples show a strong relationship between the most deteriorated areas of the paintings and higher microbial contamination.