Abstract:[Background] In view of the serious soil borne diseases and deteriorative soil physicochemical properties in protected cultivation of watermelon, we explored the effect of microbial agent on rhizosphere microbial community and soil nutrients of watermelon. [Objective] To study the effect of different treatments of Aspergillus niger agent on rhizosphere bacterial diversity and community structure, as well as soil physicochemical properties of watermelon. [Methods] Through high-throughput sequencing, we analyzed the effect of A. niger agent inoculation alone (Y), amino oligosaccharin water aqua (root irrigation) alone (A) or the combination of the two treatments (YA), on the rhizosphere soil bacterial diversity and community structure of watermelon. Analytical chemistry methods were used to detect the soil physicochemical properties and elucidate the main driving factors of their dynamic changes. [Results] The bacterial α diversity indexes of Chao 1, ACE and Shannon indices increased in Y, A, and YA treatments compared with those in the control. The dominant bacterial phyla in the rhizosphere soil of watermelon consisted of Proteobacteria, Actinobacteriota, Chloroflexi, and Acidobacteriota, among which, Proteobacteria was the most abundant, with relative abundance in the order of A (28.26%)>Y (26.74%)>YA (22.61%). The relative abundance of Gemmatimonadaceae and Nocardioidaceae increased to 4.06% and 2.43% in Y treatment, respectively, while the relative abundance of Pseudomonas was obviously improved under A treatment (P<0.05). The relative abundance of Microvirga rose in all the three different treatments in comparison with that of the control. The content of soil total nitrogen, total phosphorus, and available phosphorus significantly elevated in Y and YA treatments. RDA suggested the significant correlation of bacterial community structure with soil pH, total nitrogen, available phosphorus, and available potassium (P<0.05), and Spearman correlation analysis suggested the significant or highly significant positive correlation of relative abundance of Pseudomonadaceae with the total nitrogen, total phosphorus, available phosphorus, and available potassium. [Conclusion] Application of A. niger agent alone or in combination with amino oligosaccharin water aqua in watermelon under protected cultivation can improve bacterial diversity, abundance of beneficial bacteria, soil physicochemical properties, and soil fertility. The findings lay a basis for the development of microbial agent with A. niger and the rational application in watermelon cultivation.