Safe drinking water and microbial inactivation from surfaces and devices are among the World Health Organization’s priorities. Plasma-activated water (PAW) inactivates microorganisms mainly by producing radicals (hydroxyl radicals, superoxide, nitrogen oxide, etc.), which form secondary reactive species like nitrates, nitrites, hydrogen peroxide, etc., from the air–liquid interface, where the plasma interacts with the water. A plasma arc device for water treatment with enhanced arc length was constructed at the Andronikashvili Institute of Physics (TSU) and used in the study. PAW’s antibacterial efficacy has been evaluated against Gram-negative E. coli and remarkably stress-resistant Gram-positive B. pumilus. This study identifies reactive oxygen (hydrogen peroxide and superoxide anions) and nitrogen species (total nitrate and nitrite ions) in plasma-activated water, analyzing their potential impact on antioxidant enzyme activity and their relationships with bacterial cell viability. B. pumilus exhibits greater resistance to plasma-activated water as a disinfectant compared to E. coli. Catalase is more effective than superoxide dismutase in protecting cells from external oxidative stress, based on the two antioxidant enzymes studied.