Abstract Oxalate film formation is a pathology that often occurs in mural paintings and may result from the concomitant action of microorganisms and environmental conditions. Low Choir of the Convent of Nossa Senhora da Saudação (Portugal) has mural paintings with an extraordinary beauty, which over time have been suffered polychromy degradation and biofilm formation, presenting an ideal case study to investigate the role and impact of microorganisms in the biodeterioration process. Bacterial populations, filamentous fungi belonging to the genera Cladosporium, Penicillium, Nectria and yeast strain of the genera Rhodotorula were isolated from these wall paintings. The penetration of fungal hyphae in the microstructure of mortars, observed by scanning electron microscopy, seems to be responsible for cracking and detachments in some areas of the painting. The study revealed that the veils on the surface of the paintings are essentially oxalates and that these biofilms are caused by metabolic activity of bacterial communities. Furthermore, the colour alteration of green areas due to microorganisms was detected by Raman microscopy, in real samples and under in vitro conditions, being the result of the metabolic activity of microorganisms present on the paintings, which promote calcium oxalates formation over the malachite paint layers.