Plasma-activated water (PAW) is an innovative antimicrobial solution with great potential in enhancing food safety, advancing healthcare, and supporting environmental sustainability. This thesis investigates the inactivation mechanisms of \textit{Escherichia coli} (\textit{E. coli}) using PAW, focusing on its physicochemical properties, the role of reactive species, and their biological impacts. PAW is produced by exposing water to low-temperature plasmas, generating reactive oxygen and nitrogen species (RONS), which are central to its antimicrobial properties due to their oxidative and nitrosative stress capabilities. A novel and portable PAW reactor was developed during this thesis, enabling non-contact treatment of water substrates using a dielectric barrier discharge. \textit{In situ} FTIR spectroscopy revealed nitrogen oxides-dominated plasma chemistry, consistent with VIS-spectrophotometry characterization of PAW. The effects of operational parametersâ including water recirculation, discharge power, and pumping speedâ on PAW properties were systematically studied. Additionally, the stability of PAW was monitored over 72 hours of storage at 25°C. The relationship between these physicochemical properties and PAW antimicrobial efficacy was quantified to identify the optimal operational conditions. A multi-faceted approach was employed to investigate the mechanisms underlying \textit{E. coli} inactivation. Flow cytometry demonstrated that PAW does not induce a viable but non-culturable state but rather leads to complete cell inactivation. Scanning electron microscopy revealed damage to the outer bacterial membrane, while the absence of cytoplasmic leakage suggested a non-lytic inactivation mechanism. RNA extracted from treated cells was extensively damaged and unsequenceable. Proteomic analysis identified the downregulation of pathways related to metabolic activity and RNA synthesis, while pathways associated with ion transport, outer membrane organization, and stress responses were enriched. Upregulated proteins, such as the Na\textsuperscript{+}/H\textsuperscript{+} antiporter NhaA, and nitrite reductase NirB, highlighted bacterial efforts to restore ion balance and mitigate nitrosative stress. Overexpression of the DNA repair protein Endonuclease V (Nfi) suggested attempts to repair nitrosative DNA damage. Furthermore, proteins related to antibiotic resistance, such as AmpC and MacA, were upregulated, indicating that PAW treatment triggers broader defense mechanisms beyond immediate metabolic stress responses. These findings suggest an inactivation mechanism associated with reactive nitrogen species-induced internal cellular damage. The proposed inactivation mechanism, synthesized from all the findings in this thesis, is discussed in the concluding chapter. Single-cell time-lapse microscopy revealed a heterogeneous bacterial response to PAW treatment. A subset of \textit{E. coli} cells exhibited filamentous morphology as a transient survival strategy before returning to a bacillary shape during recovery in LB media. These observations provide crucial insights into bacterial survival strategies post-PAW treatment, essential for the safe and effective development of PAW technology. This thesis underscores the potential of PAW as a novel and eco-friendly antimicrobial solution and contributes to the broader understanding of plasma-liquid interactions, addressing critical aspects of PAW-based decontamination.