Murals are one of the important cultural heritages of mankind. The microbial control of murals is an important subject in mural painting conservation. In recent years, electron beam radiation sterilization has attracted more and more attention in the field of cultural relic protection. Murals are immovable cultural relics, so conventional electron beam irradiation equipment can not be used. However, the development of small mobile electron beam irradiation equipment shows the potential of radiation’s application in the sterilization protection of immovable cultural relics such as murals. A feasibility study of radiation sterilization in mural paintings is needed to investigate the effect of sterilization and the influence of sterilization dose on the stability of mural painting pigments and bonding materials. In this paper, the radiation effects of typical bacteria in tomb murals and mineral pigment powder in ancient Chinese paintings were studied in a laboratory. Firstly, aeromonas hydrophila (Aer.h) and penicillium flavigenum (PNC) were selected as representative strains to determine the appropriate sterilization dose for murals. Then, the effects of radiation on seven kinds of ancient Chinese mineral pigments and white calcium carbonate in the ground layer were verified. The results are as follows: the radiation dose of 10 kGy can effectively remove the two typical strains. This sterilization dose will cause a color difference in calcium carbonate and lead white, while other color pigments are essentially stable. Based on the color difference and UV-vis intensities of the four white carbonate samples, the color change in two of them increased with increasing the dose up to 30 kGy, after which signs of saturation began to appear. X-ray diffraction (XRD), Raman spectra, X-ray photoelectron spectroscopy (XPS) and X-ray absorption near edge structure (XANES) spectra showed that the chemical structure of the samples did not change after irradiation. The formation of free radicals in treated samples was confirmed using an electron paramagnetic resonance (EPR) spectrum test. According to all characterization results, the color difference between the four white carbonate samples may be due to the combination of unpaired electrons and defects in the process of electron beam irradiation to form color centers. After forming the color center, the light absorption of the four samples changed. This is a reversible change, but the samples will take a long time to return to their original state. This study focuses on the influence of electron beam radiation on pigment composition, which is a preliminary exploration of whether radiation sterilization can be applied to the protection of ancient Chinese mural paintings, and the experimental results can provide basic data for later application.