Microorganisms play a decisive role in the biological decay of building materials and works of art. Their properties, the frequent occurrence and the effects of microbial growth demand for more detailed studies in order to identify the inhabiting microbiota and to elucidate the risks for humans and materials. In the first study molecular techniques were optimized and evaluated for building biological investigations in order to find the most suitable DNA extraction method. Therefore, up to 13 DNA extraction protocols were tested with 3 typical building materials. The best overall performance showed the FastDNA Spin Kit for soil (MP Biomedicals). Furthermore, five ecological, indoor insulation systems were investigated for their bio-susceptibility against fungal growth. The biological contamination was assessed by classical cultivation- and by molecular techniques; both under laboratory- and under natural conditions. From the microbiological and hygienically point of view, the most appropriate thermal insulation system was presented by the bloated Perlite plaster. In the second study we investigated the microbiota responsible for the rosy discoloration of mural paintings in three Austrian historical buildings. Culture-dependent and independent techniques were applied for the identification of the microbes. Cloned sequences revealed the dominant occurrence of halophilic members of Actinobacteria. The majority of the colored, halophilic bacterial strains could be associated with members of the Firmicutes and representatives of the Archaea affiliated with the genera Halobacterium, Halococcus and Halalkalicoccus. A high similarity was observed among the microbiota detected in the three buildings as well as to other reported historical sites. Furthermore, under laboratory conditions, two selected strains proved that these microorganisms were responsible for the rosy pigmentation and biodeterioration on the ancient paintings.