Gamma radiation is an effective technique for the conservation of art collections, reducing microbial loads and can be obtained by the emission of a radioactive isotope, such as Cesium 137. A Portinari’s artwork (National Museum, Brazil) was analysed, and the fungi contained therein were isolated and treated with gamma radiation for decontamination. Radiation doses used were 16, 19 and 22kGy. Results indicated 11 genera and 17 species of fungi isolated. Penicillium and Cladosporium were isolated from air, the artwork, and its support. The genera Penicillium, Cladosporium, Nigrospora and Curvularia showed high resistance to radiation (16kGy) being the most resistant species Cladosporium, with no growth just under 22kGy. The results outlined indicate that the rates of DNA damage and repair were critical, depending on chronic or acute doses. The biochemical mechanism acting on fungal cells under irradiation was basically the inactivation of specific enzymes and, probably, DNA damage, particularly stimulating double-strand breaks.