Excessive use of chemical fertilizers in agriculture has become a major global source of pollution, leading to issues such as soil compaction, reduced fertility, eutrophication of water bodies, and air pollution. To address these challenges, the application of biofertilizers, such as rhizobial inoculants, has gradually become an effective, low-cost, and sustainable solution. In this study, the variety Trifolium ambiguum Bieb. (Mengnong clover No. 1) was used as the test material, and two rhizobial strains (R1 and R2) were employed for field inoculation trials. In April 2022, Caucasian clover was planted in an experimental field at Inner Mongolia Agricultural University. Each plot measured 3 m × 4 m and was arranged in a completely randomized design with three replications. In the regreening stage of 2023, rhizobial inoculation treatments were applied, with a control group included for comparison. This research examined the effects of rhizobial inoculation on the growth indicators of Caucasian clover, soil physicochemical properties, soil enzyme activities, and soil microbial communities. The results showed that rhizobial treatment increased the plant height and yield of Caucasian clover, improved soil physicochemical properties and enzyme activities, and positively affected soil microbial diversity and abundance. These changes enhanced soil fertility and optimized microbial community structure, promoting plant growth. The inoculation effect of strain R1 was superior to R2. In conclusion, rhizobial inoculants R1 and R2 can serve as effective biofertilizers for agricultural production.