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Over time, the proportion of resistant bacteria will increase. This is a major concern. Therefore, effective and biocompatible therapeutic strategies against these bacteria are urgently needed. Non-thermal plasma has been exhaustively characterized for its antibacterial activity. This study aims to investigate the inactivation efficiency and mechanisms of plasma-generated nitric oxide water (PG-NOW) on pathogenic water, air, soil, and foodborne Gram-negative and Gram-positive bacteria. Using a colony-forming unit assay, we found that PG-NOW treatment effectively inhibited the growth of bacteria. Moreover, the intracellular nitric oxide (NO) accumulation was evaluated by 4-amino-5-methylamino-2′,7′-dichlorofluorescein diacetate (DAF-FM DA) staining. The reduction of viable cells unambiguously indicates the anti-microbial effect of PG-NOW. The <i>soxR</i> and <i>soxS</i> genes are associated with nitrosative stress, and <i>oxyR</i> regulation corresponds to oxidative stress in bacterial cells. To support the nitrosative effect mediated by PG-NOW, we have further assessed the <i>soxRS</i> and <i>oxyR</i> gene expressions after treatment. Accordingly, <i>soxRS</i> expression was enhanced, whereas the <i>oxyR</i> expression was decreased following PG-NOW treatment. The disruption of cell morphology was observed using scanning electron microscopy (SEM) analysis. In conclusion, our findings furnish evidence of an initiation point for the further progress and development of PG-NOW-based antibacterial treatments.