Biochar can enhance organic carbon storage and mitigate the adverse effects of pesticides in the soil. However, the mechanisms by which field-aging affects the impacts of biochar on herbicide behavior and the composition of microbial communities in the soil remain unclear. This study aimed to investigate the influences of aged and fresh biochar on herbicide behavior and microbial community structure in the soil. Herein, with 14C-labeled technology, aged treatment (soil amended with field-aged biochar), fresh treatment (soil amended with fresh biochar), and control (soil without biochar) were installed to evaluate their treatment capacities. The results showed that the average leaching out and mineralization of simazine in the aged treatment were significantly higher by 4.8% and 1.66% (P < 0.05) compared with the fresh treatment. Relative to the control, the pesticide was significantly adsorbed (P < 0.05) in the aged treatment. The abundance of arbuscular mycorrhizal fungi (AMF) significantly increased by 1.03 and 1.16-fold, whereas fungi increased dramatically by 1.02-fold and decreased by 1.21-fold in the aged and fresh treatments, respectively (P < 0.05). In addition, eukaryotes were effectively reduced by 1.02 and 1.14-fold in these treatments, respectively (P < 0.05). This study suggests that field aging can undermine the impacts of biochar on pesticides and modify the microbial community structure in the soil environment.