Background <p><i>Escherichia coli</i> (<i>E. coli</i>) is a major foodborne pathogen that affects 2.8&#xa0;million people globally. The widespread use of antibiotics and disinfectants in livestock production has contributed to the emergence of multidrug-resistant strains, underscoring the need for resistance surveillance and the development of alternative control strategies, such as ozone nano water (ONW). ONW has emerged as a promising disinfectant, particularly for controlling microbial contamination in food safety and medical applications. In this study, we evaluated the bactericidal activity of ONW against four <i>E. coli</i> strains, including three clinical isolates with multidrug-resistant (MDR) phenotypes, and assessed potential short-term effects on antimicrobial susceptibility.</p> Results <p>ONW was physicochemically characterized (nano bubble size distribution, zeta potential, and stability under varied conditions). ONW caused rapid inactivation of all strains and markedly suppressed biofilm formation. Flow cytometry and microscopy (SEM and TEM) demonstrated that ONW induced significant cell membrane damage, resulting in bacterial death. Confocal Raman microspectroscopy revealed decreases in spectral features associated with membranes, cell walls, nucleic acids, and ATP, supporting a multi-target oxidative injury mechanism. Interestingly, ONW did not significantly alter the antimicrobial sensitivity of <i>E. coli</i> strains across three consecutive generations, suggesting its potential as a sustainable disinfectant without promoting resistance.</p> Conclusion <p>These results indicate that ONW is an effective and potentially sustainable disinfectant against <i>E. coli</i>, including MDR strains, with applicability to medical and agricultural settings while minimizing concerns about resistance selection.</p>