Antimicrobial resistance continues to be a rising global threat to public health. It is well recognized that wastewater treatment plants are reservoirs of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). However, traditional disinfection techniques are not effective to simultaneously remove ARB and ARGs, and the dynamic analysis of ARB inactivation have also been deficient. In this study, sulfidated nano zerovalent iron (S-nZVI) coupled with persulfate (PS) was applied to simultaneously remove both ARB (E. coli K-12 with RP4 plasmid) and ARGs (extra- and intracellular ARGs). S-nZVI/PS completely inactivated ARB (7.8-log reduction) within 10 min and degraded all extracellular ARGs (8.0-log reduction) within 5 min. These efficiencies were significantly higher (decay rate constant, k = 0.138 min) than those achieved individually (S-nZVI: k = 0.076 min; PS: k = 0.008 min), implying a synergistic effect between S-nZVI and PS against ARB and ARGs. The efficient removal rate of ARB was also supported by confocal microscopy and microfluidics at a single-cell level. The complete inactivation of ARB by S-nZVI/PS was also demonstrated in real drinking water and real wastewater effluent that contained natural organic matter and suspended solids. Regrowth assays showed that the treated ARB was not observed after 72 h or longer incubation, suggesting that ARB was permanently inactivated by radicals such as SO and •OH. The destruction of bacterial cells compromised the removal efficiency of the intracellular ARGs, with only ~4.0-log reduction after 60 min treatment by S-nZVI/PS. Collectively, our results suggest the feasibility of S-nZVI coupled with PS for simultaneous ARB and ARGs removal in real water matrices.