Mural paintings are some of the oldest and most important cultural expressions of mankind and play an important role for the understanding of societies and civilizations. These cultural assets have high economic and cultural value and therefore their degradation has social and economic impact. The present work presents a novel microanalytical approach to understand the damages caused by microbial communities in mural paintings. This comprises the characterization and identification of microbial diversity and evaluation of damage promoted by their biological activity. Culture-dependent methods and DNA-based approaches like denaturing gradient gel electrophoresis (DGGE) and pyrosequencing are important tools in the isolation and identification of the microbial communities allowing characterization of the biota involved in the biodeterioration phenomena. Raman microspectrometry, infrared spectrometry, and variable pressure scanning electron microscopy coupled with energy-dispersive X-ray spectrometry are also useful tools for evaluation of the presence of microbial contamination and detection of the alteration products resulting from metabolic activity of the microorganisms. This study shows that the degradation status of mural paintings can be correlated to the presence of metabolically active microorganisms.