Background and aims <p>Pepper (<i>Capsicum</i> ssp.) is a widely planted vegetable crop, but persistent planting results in soil continuous cropping obstacles, negatively affecting pepper plant development and productivity. To address this issue, natural soil biotin (NSB), a novel compound microbial fertilizer, was introduced as a base fertilizer during the colonization of pepper 'Hang Jiao Zao Xiu'. We explored the effects of NSB on soil and pepper growth in this study.</p> Methods <p>Studies were conducted to investigate the impact of NSB on pepper growth by comparing several agronomic traits of pepper plants between the NSB treatment group and the control group. Metagenomic sequencing was used to analyze the effects of NSB on rhizosphere soil microorganisms. And the relationship among rhizosphere soil microorganisms, soil pH, and pepper plant traits was also analysed.</p> Results <p>The findings revealed that NSB treatment significantly increased pepper plant height, breadth, stem diameter, dry matter content, yield per plant, and the amounts of crude fiber and capsaicin in fruits. Additionally, NSB application slowed the soil pH drop, altered the microbial dominant population structure, and reduced the number of harmful microorganisms in the rhizosphere soil of pepper plants. Correlation analysis indicated that some important agronomic traits such as yield, capsaicin content, and total root surface area were correlated with the relative abundance of some microbial dominant populations.</p> Conclusions <p>Therefore, using NSB may improve the soil environment, regulate soil pH and microbial community structure, promote pepper plant root growth, and ultimately enhance pepper plant growth, fruit yield, and quality.</p>