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The chemistry, antimicrobial efficacy and energy consumption of plasma-activated water (PAW) was optimized by altering the discharge frequency, ground-electrode configuration, gas flow rate and initial water conductivity for two reactor configurations, i.e., air pin-to-liquid discharge and air plasma-bubble discharge in water. The ratio of NO 2 − and NO 3 − formation was altered to optimise the antimicrobial effects of PAW, tested against two Gram-negative bacteria. An initial solution conductivity of 0.2 S·m−1 and 2000-Hz discharge frequency with the ground electrode positioned inside the pin reactor showed the highest antimicrobial effect resulting in a 3.99 ± 0.13-log 10 reduction within 300 s against Escherichia coli and 5.90 ± 0.24-log 10 reduction within 240 s for Salmonella Typhimurium. An excellent energy efficiency of reactive oxygen and nitrogen species (RONS) generation of 10.1 ± 0.1 g·kW−1·h−1 was achieved. Plasma-activated water (PAW) is deemed as an eco-friendly alternative to chemical disinfection because its bactericidal activity is temporary. Optimizing the design and operation of PAW reactors to achieve high inactivation rates of more than 5-log 10 reductions, as demonstrated in this work, will support the industrial application of this technology and the scaleup at industrial level. • Increasing discharge frequency from 1000 to 2000 Hz improved the antimicrobial characteristics of PAW by the pinreactor • Increasing initial liquid conductivity in the pin reactor increased the bacterial inactivation to 1.74–4.4-log 10 reduction • Ground electrode inside the pin reactor caused higher E. coli (3.99-) and S. Typhimurium (5.90-log 10 reduction) inactivations • Altering ground-electrode position from inside to outside the bubble reactor changed the NO 2 −/ NO 3 − ratio from 0.92 to 2.69 • The optimal PAW configuration, achieving more than 5-log reduction of S. Typhimurium , was the pin-to-liquid discharge [ABSTRACT FROM AUTHOR]