Murals are a significant cultural heritage of humanity, and one of the conservation studies is to control the growth of microorganisms. General biocide agents can be used to preserve murals while also providing new organic carbon sources and increasing environmental pollution. In recent years, radiation technology has shown promising prospects for use in heritage protection. Five microorganisms often found in murals were irradiated with an electron beam in this study, and six mineral pigments were tested for color change, Raman spectra and pigment layer cohesion after irradiation. The result showed that irradiation at 20 kGy can basically eliminate Pseudomonas citronellolis, Bacillus sporothermodurans, Streptomyces vinaceus, and Streptomyces griseolus from the culture medium, but only inhibited the growth of Penicillium flavigenum. Lead white pigment showed a color difference of 5.56 (∆E*97) after irradiation, but lead tetroxide, azurite, malachite, ferrous oxide, and cinnabar showed no visible changes. The Raman spectra of the irradiated and unirradiated samples were basically the same. E-beam radiation did not affect the surface cohesion of the pigment layer. This preliminary work shows the potential of electron-beam technology in mural protection and provides basic research and relevant experience for the subsequent in situ mural protection work.