The inventory of microorganisms responsible for biological deterioration of ancient paintings has become an integral part of restoration activities. Here, the microbial agent of rosy discoloration on medieval frescoes in the Crypt of the Original Sin (Matera, Italy) was investigated by a combination of microscopic, molecular and spectroscopic approaches. The bacterial community from three rosy-discoloured painting sites was characterized by 16S rRNA gene-based techniques. The eubacterial population was prevalently composed of Actinobacteria, among which Rubrobacter radiotolerans-related bacteria accounted for 63-87% of the 16S rRNA gene pool per sampled site. Archaea, with prevalence of Haloarchaea-related species, were detected in one of the three sites where they accounted for < 0.1% of the total 16S rRNA gene pool. Raman spectroscopy confirmed the identity between R. radiotolerans carotenoids (bacterioruberins) and pigments responsible for colour alteration of frescoes. This investigation provides the first evidence of a causal relationship between heavy contamination by Rubrobacter-related bacterioruberin-producing bacteria and rosy discoloration of ancient wall paintings.