It is widely known that antibiotics can affect the structure and function of soil microbial communities, but the specific degree of impact and controlled factors on different indicators remain inconclusive. We conducted a multiple hierarchical mixed effects meta-analysis on 2564 observations that were extracted from 60 publications, to comprehensively assess the impact of antibiotics on soil microbiota. The results showed that antibiotics had significant negative effects on soil microbial biomass, α-diversity and soil enzyme activity. Under neutral initial soil, when soil was derived from agricultural land or had a fine-textured, the negative impacts of antibiotics on soil microbial community were exacerbated. Both single and mixed additions of antibiotics had significant inhibitory effects on soil microbial enzyme activities. The Random Forest model predicted the following key moderators involved in the effects of antibiotics on the soil microbiome, and antibiotics type, soil texture were key moderators on the severity of soil microbial biomass changes. Soil texture, temperature and single or combined application constitute of antibiotics were the main drivers of effects on soil enzyme activities. The reported results can be helpful to assess the ecological risk of antibiotics in a soil environment and provides a scientific basis for the rational of antibiotics use in the soil environment.