The chiral herbicide imazethapyr (IM) is frequently used to control weeds in soybean fields in northeast China. However, the impact of IM enantiomers on microbial communities in soil is still unknown. Genetic markers (16S rRNA V3-V4 regions) were used to characterize and evaluate the variation of the bacterial communities potentially effected by IM enantiomers. Globally, the bacterial community structure based on the OTU profiles in (-)-R-IM-treated soils was significantly different from those in (+)-S-IM-treated soils, and the differences were enlarged with the treatment dose increasing. Interestingly, the Rhizobiaceae family and several other beneficial bacteria, including Bradyrhizobium, Methylobacterium, and Paenibacillus, were strongly enriched in (-)-R-IM treatment compared to (+)-S-IM treatment. In contrast, the pathogenic bacteria, including Erwinia, Pseudomonas, Burkholderia, Streptomyces, and Agrobacterium, were suppressed in the presence of (-)-R-IM compared to (+)-S-IM. Furthermore, we also observed that the bacterial community structure in (-)-R-IM-treated soils was more quickly restored to its original state compared with those in (+)-S-IM-treated soils. These findings unveil a new role of chiral herbicide in the development of soil microbial ecology and provide theoretical support for the application of low-persistence, high-efficiency, and eco-friendly optical rotatory (-)-R-IM.