This work aimed to evaluate the effectiveness and long-term performance of mold disease control methods during relocation of murals from the tomb of Han Xiu, and to study the causes of mold diseases as well as the prevention and control measures used in the current restoration stage. The characteristics of mold diseases were analyzed using ultra depth-of-field microscopy. The community composition of fungi was identified using molecular techniques. In addition, the influences of mold control methods during relocation on the development and succession of microflora in the restoration stage was explored by combining the mold identification results in the relocation stage and the monitoring data of the preservation environment. The following results were obtained:the 11 strains isolated from the mold samples in the restoration stage belonged to Penicillium and Cladosporium; compared with the relocation stage, the diversity of molds on the mural surfaces was significantly reduced, and the composition of the dominant fungus Penicillium was also significantly different. The results also show the effective inhibition by clotrimazole and other anti-mold agents on the growth of Aspergillus, Fusarium and Alternaria, and the adaptability and selectivity of Penicillium fungi to the environment. The action of mold metabolites on the mural body caused mildew pollution, discoloration, disruption and other diseases of the murals. In addition, Penicillium mycelium had deeply entered into the interior, causing great harm to the stability of the mural structure. Therefore, it is recommended to classify and clean the yellow and black molds on the surfaces, screen mildew prevention agents and develop environmental control of exhibition cases promptly, to achieve long-term prevention and control of mold diseases of the murals from the tomb of Han Xiu.