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The need for sustainable and reliable decontamination methods is driven by concerns regarding antibiotic resistance, as well as environmental and cost-efficiency challenges associated with traditional methods. Plasma-activated water (PAW) holds significant promise as an innovative and eco-friendly decontamination method. Nevertheless, prior to industrial implementation of PAW-based decontamination devices, a deep understanding of the bacterial inactivation mechanisms and its interplay with PAW chemical composition is required. Advancing in this field requires interdisciplinary and collaborative research using standardized practices with cost-effective and shareable PAW reactors that are still missing today. Here, to this end, a portable PAW reactor, featuring a surface dielectric barrier discharge (SDBD) that operates in air, is presented. The gaseous and aqueous phases were chemically characterized by Fourier transform infrared (FTIR) absorption spectroscopy and vis-spectrophotometry, respectively. To assess PAW antimicrobial efficacy Escherichia coli was employed as a model organism on six different PAW samples for three different treatment times. The significance of water recirculation in controlling the chemical composition of PAW, altering the nitrogen species balance in favor of NO2−, is demonstrated. This chemical modification consequently enhanced the antimicrobial effectiveness of PAW, reaching a log reduction of ∼6. Exposing E. coli to equivalent concentrations of RONS to those found in PAW attained similar log-reductions, indicating that acidified nitrites are key to PAW antimicrobial activity. Through an innovative and portable design, this study illustrates the crucial role of water recirculation in tailoring PAW composition, leading to improved decontamination capabilities and establishing a key parameter for further optimization of PAW production.