Plasma-activated water (PAW), generated through Surface Dielectric Barrier Discharge, was tested against microbial contaminants. We assessed how the time of exposure to plasma treatment and the gas flow rate impact the chemical composition of PAW, and, in turn, how it influences these factors influence its efficacy against microorganisms. The effectiveness of PAW treatments was evaluated against the fungal phytopathogen Botrytis cinerea and both pathogenic (Xanthomonas campestris pv. vesicatoria) and beneficial (Bacillus amyloliquefaciens) bacteria. The physicochemical properties of PAW were assessed as the concentration of reactive species, namely, NO