Background: The deposition of the airborne fungi onto cultural heritage is associated closely with the subsequently biodeterioration taking place. In this study, a systematic survey of the culturable airborne fungi was carried out in the occurrence environments of the wall paintings that preserved in the Tiantishan Grottoes and the Western Xia Museum, China. Bio-aerosol sampler was used for sampling in four seasons of 2016. Culture-dependent and -independent methods was taken to acquire the airborne fungal concentration and purified strains; by the extraction of genomic DNA, amplification of fungal ITS rRNA gene region, sequencing, and phylogenetic analysis, thereafter the fungal community composition and distribution characteristics of different study sites were clarified. Finally, we disclosure the main environmental factors which may responsible for the dynamic changes of the airborne fungi at the sampling sites. Results: The concentration of culturable airborne fungi was in a range from 13 to 1576 CFU/m3, no significant difference between the two sites at the Tiantishan Grottoes, with the obvious characteristics of the seasonal variation, in winter and spring were higher than in summer and autumn. Meanwhile, a significance difference in fungal concentration between inside and outside of the Western Xia Museum, the outside of the museum was far more than the inside of the museum in the four seasons, particularly in the winter. Eight fungal genera were detected, including Cladosporium, Penicillium, Alternaria, and Filobasidium as the dominant groups. The airborne fungal community structures of the Tiantishan Grottoes showed a distinct characteristic of seasonal variation and spatial distribution. Relative humidity, temperature and seasonal rainfall all have influences on the airborne fungal distribution. Some of the isolated strains have potential to cause biodeterioration of the ancient wall paintings. Conclusions: The prominent seasonal variation of airborne fungal concentrations, community structures and distribution patterns were found in this study, that have close relationship with local climate conditions of the Tiantishan Grottoes. Therefore, it is necessary to carry out the long period monitoring of airborne fungal flora in the future, and integrated it into a pre-warning monitoring and conservation system.