Biochar has great potential in reducing the abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) from soil. However, its efficiency in removing other biological pollutants, such as human bacterial pathogens (HBPs) and virulence factor genes (VFGs), is rarely studied. Herein, by pyrolyzing rice straw (RS) and pine wood (PW) at 350 and 700 °C, we prepared a series of biochar (RS350, RS700, PW350, and PW700) and investigated their impacts on the abundance and pathogenicity of HBPs. Compared with PW biochar, RS biochar effectively reduced the abundance of HBPs by 6.3−40.1%, as well as their pathogenicity, evidenced by an 8.2− 10.1% reduction in the abundance of VFGs. Mechanistically, more persistent free radicals (PFRs) were formed in RS biochar than that of PW biochar during pyrolysis, and PFRs triggered the degradation of N-butyryl-L-homoserine lactone (C4-HSL) from 1.05 to 0.68 ng/kg, thereby disturbing the quorum sensing (QS) of HBPs. Once the QS was disturbed, the communications among HBPs were hindered, and their virulence factors were reduced, which ultimately lowered the abundance and pathogenicity of HBPs. Collectively, our study provides insights into the role of biochar in decreasing the risks of HBPs, which is significant in the development of biochar-based technologies for soil remediation.