Abstract: The continuous monoculture problem commonly causes huge economic losses in modern agricultural production. The occurrence and development of continuous cropping obstacles pose a serious threat to the yield and quality of Chinese medicinal plants; therefore, it is important to explore effective strategies to alleviate the continuous cropping obstacles. In this study, we evaluated the effects of microbial fertilizer application on the continuous cropping obstacles and pharmacological activities of radix pseudostellariae (Pseudostellaria heterophylla). The effects of microbial fertilizer application on the yields of continuous monocultured radix pseudostellariae for 4 years under field localization experiments were investigated. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography-mass spectrometry (HPLC-MS) were used to analyze the effects on the rhizosphere soil microorganisms and the main effective components of radix pseudostellariae. We also used pharmacological tests to analyze the efficiency of radix pseudostellariae under different treatments. Our results showed that microbial fertilizer significantly improved the growth of continuous monocultured radix pseudostellariae, and the yield increased by 68.28%, 111.58%, 257.54%, and 133.23% in 4 years, respectively. Microbial fertilizer application significantly increased the abundance of beneficial Pseudomonas spp., decreased pathogenic Fusarium oxysporum in the rhizosphere soil, and increased the contents of total polysaccharides and heterophyllin B in radix pseudostellariae as compared with those of the consecutive monoculture treatments. The types and contents of amino acids in radix pseudostellariae under microbial fertilizer application were similar to those in the 1-year monoculture and standard radix pseudostellariae. Eight types of amino acids in the microbial fertilizer amendment and the newly planted radix pseudostellariae were not significantly different. Pharmacological tests showed that radix pseudostellariae alleviated the damage caused by cyclophosphamide in mice. There were no significant differences in the spleen index, liver weight, epididymal fat weight, and white or red blood cell content of mice treated with plants of the microbial fertilizer amended and newly planted radix pseudostellariae. The effect of radix pseudostellariae on platelet recovery of mice under microbial fertilizer amendment was consistent with that of standard radix pseudostellariae. Moreover, the total antioxidant capacity of radix pseudostellariae under microbial fertilizer amendment was stronger, and the superoxide dismutase content was significantly higher than that of newly planted and standard radix pseudostellariae. Compared with newly planted radix pseudostellariae, the microbial fertilizer amendment significantly increased the expression of immune factors IL-2 and IFN-r in the liver tissue of mice. There was no significant difference in the expression of TNF-α between the microbial fertilizer amendment and newly planted radix pseudostellariae. In conclusion, microbial fertilizer amendment effectively alleviated the continuous cropping obstacle and improved the quality and pharmacological activities of radix pseudostellariae. Our results provide guidance for studying alleviation strategies in other Chinese medicinal plants under consecutive monoculture regimes and for evaluating the quality and pharmacological activities of modified medicinal plants.