Abstract: To address the issue of root rot prevention Astragalus membranaceus var. mongholicus , this study conducted a study and test on the effect of microbial agents on root rot control efficiency, which has significant practical significance for reducing the incidence of root rot in repeatedly cropped Astragalus membranaceus var. mongholicus root rot and enhancing yield and quality. In this study, three biological bacteriological agents-Bacillus subtilis, Trichoderma Harzianum, and Bacillus amyloliquefaciens were used in Weiyuan County under controlled experimental conditions. An orthogonal dip root test design labeled '3414' was employed, incorporating three factors and four levels. Through the assessment and analysis of emergence rate , root rot control efficacy, soil enzyme activity, soil physicochemical property and agronomic trait indices, the results were used to determine the optimal timing and dosage for the application of these biological agents. Results indicated that the HQ-13 microbial agent combination (60.0 g/mU B. subtilis + 50.0 g/mU T. harzianum + 27.5 g/mU B. amyloliquefaciens) exhibited the most favorable effect in enhancing emergence, soil enzyme activity, soil physicochemical property, and reducing root rot control. The HQ-10 microbial agent combination (90.0 g/mU B. subtilis + 100.0 g/mU T. harzianum + 55.0 g/mU B. amyloliquefaciens) root dipping treatment shows the best performance in improving agronomic traits, increasing soil enzyme activity, and improving soil physicochemical properties and chemical indicators of Astragalus membranaceus var. mongholicus. Correlation analysis data showed that the root rot control effect was positively correlated with seedling emergence and agronomic traits (P  <  0.05), and negatively correlated with soil enzyme activity and soil physicochemical property (P  <  0.01). Therefore, the author suggested that the HQ-13 microbial combination dip root treatment should be used at the emergence stage of Astragalus membranaceus var. mongholicus and the HQ-10 microbial combination should be used during the growth stage.