It is important to characterize the microorganisms involved in biodeterioration processes to understand their effects on cultural assets and to define an efficient strategy for protecting artworks, monuments, and buildings from microbiological recolonization. In this study, we analyzed the microbial communities dwelling on the verso (front) and recto (back) sides of a 17th century easel painting attributed to Carlo Bononi, an Italian artist of the first Baroque period. Cultivable bacteria and fungi colonizing the painting were isolated and identified in order to characterize the microbial community possibly involved in deteriorating the pictorial layer of the painting. The isolated bacterial strains belonged to the Staphylococcus and Bacillus genera. Furthermore, culture-dependent techniques and SEM/EDS analyses revealed the presence of filamentous fungi of the genera Aspergillus, Penicillium, Cladosporium, and Alternaria. The chemical compositions of pigments were consistent with typical 17th century paintings, and some of the identified pigments, namely red lac and red and yellow earths, could be exploited as nutrient sources by painting-associated microorganisms. The study also evaluated, in vitro, the potential decontaminating activity of a biocompound, containing spores of Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium. The results indicated the ability of this biocompound to counteract the growth of contaminating microorganisms that are potentially dangerous to the painting, suggesting the potential use of these microorganisms to prevent biodeterioration of artworks.