Pathogenic microorganisms pose a global threat to public health and environment. Common antibacterial chemicals produce toxic residues, inevitably harming the environment. Electrolyzed oxidizing water (EOW), a promising environment-friendly alternative disinfectant, still lacks effective production processes, sufficient bactericidal efficacy and stability, while the enabling physico-chemical mechanisms remain unclear. Here, we report, for the first time, an effective hybrid plasma electrochemical EOW production process and reveal the mechanisms by combining nonthermal plasmas and a two-chamber electrochemical cell separated by a cation exchange membrane (CEM) for decoupling the chemical reactions during the plasma treatment of water. Experimental results demonstrate that combined chlorine (chloramine) was the main chlorine product in the plasma-enhanced EOW (P-EOW) without a membrane, owing to the consumption of free chlorine (Cl2, HOCl, ClO−) by plasma-generated reactive nitrogen species. With a CEM in the plasma electrolysis system and through controlling the plasma discharge polarity, the production of free chlorine and other reactive species can be selectively controlled, with the highest concentration of free chlorine obtained in the negative plasma-enhanced EOW (NP-EOW). According to the transportation of cations by the CEM, the high concentrations of free chlorine may be attributed to the higher consuptions of H+ in cathode cell of negative plasma. The study of antibacterial ability of EOW produced under different conditions revealed that Staphylococcus aureus cells were best inactivated by the NP-EOW with CEM, which is mainly attributed to the higher concentration of free chlorine. This study demonstrates the feasibility of plasma-enhanced microbial electrolytic disinfection and offers new insights into the fundamental aspects of P-EOW chemistries for the future development of sustainable, efficient, and cost-effective multipurpose sustainable chemical technologies for water research and treatment.