Objective: To reveal the changes of fungal community structure and diversity in walnut the potential health threats during natural mildew through studying the fungal infection process of walnut during natural mildew. Methods: The fungal ITS rDNA of fresh walnut kernel samples with different mildew time was sequenced by Illumina Miseq high-throughput sequencing technique， and the changes of fungal community composition and diversity in the process of fresh walnut kernel mildew were analyzed. Results: Ascomycota is the main fungal species in the mildew process of walnut samples， with a relative abundance of 27.41%-99.98%， followed by Basidiomycota with a relative abundance of 0.01%-32.56%. At the fungal level， it was found that the main dominant populations in the mildew process were Fusarium， Alternaria， Gibberella and Aspergillus. The mildew process of walnut in a week can be divided into three periods: Period Ⅰ (0-2 days)， Period Ⅱ (3-4 days) and Period Ⅲ (5-7 days). In stages of Period Ⅰ and Period Ⅱ， Fusarium was the dominant genus， and the main fungus that change significantly among other periods were Fusarium， Aspergillus， Alternaria， Gibberella and Penicillium. Conclusion: The changes of fungal microbial community during mildew of walnut could be divided into three periods， during which the fungal diversity showed a downward trend， and there were a variety of harmful pathogens， such as Fusarium and Aspergillus， etc.， which might cause potential safety risks if not handled properly.