Biochar-supported ZVI have received increasing attention for their potential to remove nitrobenzene in groundwater and soil. However, the capacity of this material to enhance the biological reduction of nitrobenzene and alter microbial communities in anaerobic groundwater have not been explored. In this study, the nitrobenzene removal performance and mechanism of modified biochar-supported zerovalent iron (ZVI) composites were explored in anaerobic soil. The results showed that the 700 °C biochar composite enhanced the removal of nitrobenzene and inhibited its release from soil to the aqueous phase. NaOH-700-Fe50 had the highest removal rate of nitrobenzene, reaching 64.4%. However, the 300 °C biochar composite inhibited the removal of nitrobenzene. Microbial degradation rather than ZVI-mediated reduction was the main nitrobenzene removal pathway. The biochar composites changed the richness and diversity of microbial communities. ZVI enhanced the symbiotic relationship between microbial genera and weakened competition between soil microbial genera. In summary, the 700 °C modified biochar composite enhanced the removal of nitrobenzene by increasing microbial community richness and diversity, by upregulating functional genes, and by promoting electron transfer. Overall, the modified biochar-supported ZVI composites could be used for soil remediation, and NaOH-700-Fe50 is a promising composite material for the on-site remediation of nitrobenzene-contaminated groundwater.