Abstract: In order to clarify the microbial dynamics and diversity during the secondary fermentation of Agaricus bisporus cultivation substrate, the high-throughput sequencing technology was used to determine the changes of fungal community structure during the secondary fermentation of Agaricus bisporus cultivation substrate, and to explore the relationship between community composition and environmental factors. The results showed that the total number of fungal taxonomic units (OTUs) in the four fermentation stages of the secondary fermentation process of Agaricus bisporus cultivation substrate was 999, including 10 phyla, 26 classes, 56 orders, 126 families, 240 genera, and 403 species. Among them, the species of Streptococcus thermophilus (12.72%−42.63%), unclassified_f_ Sortariaceae (0.12%−31.63%), Candida (1.30%−17.32%), Aspergillus (2.57%−8.98%), Penicillium (2.88%−9.76%), and Kernia (0.89%−4.18%) occupied the dominant position. During the fermentation process, the abundance of fungi belonging to genera such as Streptococcus thermophilus, Candida, Aspergillus, and Penicillium increased at the early and middle stages of fermentation, and then decreased at the later stage. The relative abundance of Streptococcus thermophilus and Penicillium reached the peak in the middle stage of fermentation. Ammonia emission and pile temperature were the key indicators of fermentation end point. The correlation between fungal community and ammonia emission and pile temperature was analyzed by using the redundancy analysis. The results showed that the abundance of Streptococcus thermophilus, Penicillium and Candida was positively correlated with the fermentation temperature and ammonia emission. The higher the abundance was, the shorter the secondary fermentation cycle was and the better the fermentation quality was.