Abstract: The soil-borne disease caused by changes in soil microbial flora is an important factor restricting the healthy development of the watermelon industry. We examined the responses of soil fungal communities to biological fumigation combined with microbial agents by setting up different treatments, namely a control (CK), biological fumigation (R), and biological fumigation combined with microbial agents (RB) in a two-year continuous cropping watermelon field. On the basis of soil chemical property analysis and Illumina HiSeq high-throughput sequencing technology, the changes in the composition and diversity of soil fungal communities and their relationships with soil environmental factors were examined. The results showed that compared with CK, both R and RB increased the content of available nitrogen, available phosphorus, available potassium, total nitrogen, and organic matter in soil, while reducing soil pH. Moreover, the total nitrogen content was significantly (P<0.05) higher in R treatment compared to the other treatments. The alpha indexes of fungal community showed that R and RB treatments significantly decreased the abundance and diversity of soil fungal community and were in the order of R<RB<CK. When compared with CK, the ACE indexes of R and RB decreased by 4.86% and 3.15% and the Shannon index decreased by 12.65% and 10.88%, respectively, while the Chao1 and Simpson index showed no significant difference, it also decreased by varying degrees. A total of 794 operational taxonomic units were obtained from three watermelon soil samples, which included some unidentified or unknown fungi. In the identified fungal communities, Ascomycota, Basidiomycota, and Mortierellomycota were the dominant fungi phylums, contributing to 95.14%–96.17% of the total abundance, with Ascomycota displaying the highest relative abundance. The relative abundance of Ascomycota and Basidiomycota increased under R treatment, while RB treatment promoted an increase in the relative abundance of Ascomycota and a significant decrease in the relative abundance of Basidiomycota. At the family level, Chaetomiaceae was the dominant fungi, and R and RB treatments significantly increased the relative abundances of Chaetomiaceae and Microascaceae. In addition, RB treatment generated a significantly increased relative abundance of Nectriaceae and reduced relative abundance of Helotiaceae. Cluster analysis showed that the fungal community structures of RB and CK were significantly different from that of R treatment, while the differences between RB and CK were small. RDA analysis showed that there was a correlation between soil chemical properties and soil fungal abundance after bio-fumigation combined with microbial agents; moreover, soil organic matter, total nitrogen, available potassium, and pH were the important driving factors for the changes in soil fungal community structure. In conclusion, biological fumigation combined with microbial agents can improve soil quality, and compared with biological fumigation alone, it can also improve the abundance and diversity of soil fungal communities, as well as adjust the structure of the soil fungal community in a beneficial manner. This study provides effective measures for alleviating or overcoming the obstacles associated with watermelon continuous cropping and an important theoretical basis for a healthy and sustainable development of the watermelon industry.