This work presents the application of some molecular biology techniques with high interest in cultural heritage field. These methodologies have proved to be extremely useful to characterize microbial communities colonization and their potential involvement in deterioration processes, mainly because it become possible curried out cultural-independent studies of microorganisms in both organic or inorganic substrates. These methods can be easily suited to study the biodeterioration of cultural heritage and, in the future, their application will be essential in conservation and/or restoration projects.