[Background] Microbial fertilizers have been widely used in the cultivation of organic crops in China, and their effects on the micro-ecology in soils of organic farm still needs scientific evaluation. [Objective] The high-throughput sequencing technology can be used to analyze soil microbial community accurately. We explained effects of Bacillus subtilis microbial fertilizer on organic farm root-zone soil bacterial and fungal community in the view of structure and diversity. [Methods] Under organic and rotate farming conditions, soil genomic DNA was extracted after application of Bacillus subtilis microbial fertilizer. And PCR amplification was made to establish libraries. Then, the 16S rRNA genes V3?V4 regions of soil bacterium and fungal ITS1 regions were sequenced by Illumina high-throughput sequencing technology on MiSeq platform, and related biological analysis was conducted to explain the changes of soil bacterial and fungal diversities and structures. In addition, soil chemical properties and enzyme activities, nutritional quality of organic Chinese watermelon were determined. [Results] A total of 14 199 bacterial operational taxonomic units (OTUs) and 3 378 fungal OTUs were obtained from 6 organic Chinese watermelon root-zone soil samples. The sequencing coverage of bacterial and fungal libraries were above 98% and 99%, respectively. Bacillus subtilis microbial fertilizer would increase the soil bacterial diversity while reduce the fungal diversity to a certain degree, and increased the abundance of bacteria, but it reduced the abundance of fungi significantly (P<0.05), and reduced the number of bacterial and fungal unique OTU. Proteobacteria, Firmicutes and Actinobacteria were the dominant bacteria, and Ascomycota was dominant fungi. It increased the relative abundance of Chloroflexi and Ascomycota by 46.23% and 10.01%, respectively. And, it reduced the relative abundance of Proteobacteria and Basidiomycota by 11.14% and 74.72%, respectively. It significantly reduced the pH of soil (P<0.05). Wherein, it significantly increased the content of total amino acids and soluble solid in organic Chinese watermelon (P<0.05). [Conclusion] The abundance and diversity of soil bacteria and fungi were changed with Bacillus subtilis microbial fertilizer applied. And soil pH was reduced, nutritional quality of organic Chinese watermelon was increased also.