Background and aims Current global population growth and agricultural land resource limitations have led to intensifying conflicts between grain and fruit production. Methods We designed a potted blueberry-soybean intercropping system to evaluate its impacts on crop yield, disease occurrence, and soil microbial community composition using survey statistics, high-throughput sequencing, and correlation analysis. Results The results demonstrate that the blueberry-soybean intercropping system is a feasible solution for obtaining additional soybean yield by the filed investigation. "Blueberry-polluted soil (BPS)" sampled at a depth of 5 cm and rhizosphere soil sampled from intercropped 'Huayan 1' soybean plants (HYS) showed significantly higher fungal and bacterial diversity than control bulk soil (CK) with no cultivation history. Microbial communities and unique OTUs were differentially enriched in BPS and HYS, respectively, and the HYS effect was more pronounced through high-throughput sequencing. Correlation analysis revealed that pH, organic matter, and total N were the main factors driving soil chemistry-mediated microbial differences in the community between CK and both HYS and BPS. The significantly lower microbial abundance in BPS was likely related to N fixation, whereas significantly enriched bacteria in HYS were related to the N regulatory protein C protein family, N regulatory IIA and P-II2 proteins, N fixation regulation proteins, and other N-related functions (p < 0.05), indicating that blueberry-soybean intercropping significantly improves microbial function in the soil. Conclusion These findings demonstrate that intercropping system could improve the acidification of soil and reduce the depletion of soil functional microorganisms caused by continuous monoculture of blueberries. Also, it could help coordinated development of grain and fruit production, particularly in regions facing both food shortages and limited arable land in the world.