Plasma-activated water (PAW) is a promising disinfection strategy that generates a complex mixture of reactive oxygen and nitrogen species (ROS/RNS), including hydrogen peroxide (H2O2), nitrate (NO3−), and transient oxidants, in an acidic aqueous environment. These reactive species contribute to both immediate and extended antimicrobial activity. This study investigates how the addition of low concentrations (<100 μM) of potassium iodide (KI) enhances the bactericidal properties of spark-generated PAW by enabling the in-situ generation of reactive iodine species (RIS), particularly hypoiodous acid (HIO), under acidic conditions.KI addition (10–100 μM) led to a counterintuitive, dose-dependent increase in H2O2 concentrations, from ∼1.2 mM in PAW alone to ∼1.8 mM at 30 μM KI, possibly due to iodine-mediated catalytic effects or reduced H2O2 degradation. NO3− levels also increased by ∼17 % with increasing KI. Equivalent concentrations of H2O2 + KI failed to replicate the rapid antimicrobial activity observed in PAW + KI, which achieved complete inactivation of Escherichia coli and Listeria monocytogenes planktonic cells within 3 min, compared to over 10 min for PAW alone, indicating the involvement of additional reactive species in KI-enhanced antimicrobial activity of PAW. However, Salmonella enterica planktonic cells exhibited only partial inactivation even with KI, indicating species-specific tolerance under these conditions. 24h biofilms of L. monocytogenes and E. coli were eradicated with PAW + KI in 10 min, whereas S. enterica showed only a 2-log reduction.Scavenger assays revealed that both longer-lived species (H2O2) and shorter-lived oxidants such as singlet oxygen are essential for this enhanced killing, while ozone and superoxide appeared dispensable. These findings support a multi-step antimicrobial mechanism: (1) plasma treatment creates a low pH, H2O2-rich solution; (2) iodide is oxidised to RIS such as I3− and HIO; (3) additional PAW-derived oxidants potentiate RIS chemistry; and (4) unionised HIO diffuses across bacterial membranes to induce oxidative damage.PAW-KI remained stable for at least 14 days at 4 °C, with sustained RIS activity and minimal loss of H2O2 or NO3−, suggesting preserved antimicrobial capacity over time. The antimicrobial mechanism likely proceeds through a four-step pathway: plasma-mediated generation of H2O2 and NO3−; oxidation of I− to I2 and HIO; potentiation of RIS via PAW-derived ROS/RNS; and subsequent microbial inactivation via membrane damage.Together, these results demonstrate that PAW + KI forms a powerful, in situ RIS-generating system, offering a residue-minimising and environmentally sustainable disinfection platform. Its rapid action, scalability, and reliance on only air, water, electricity, and GRAS-listed KI make it an attractive intervention for food safety, clinical disinfection, and decentralised sanitation settings.