Since high microbiological loads in water pipes of medical facilities and dental units pose a risk for human health, the establishment of bactericide agents should be advanced to minimize the contamination level. We focused on the efficacy of plasma-activated water (PAW) as a novel disinfectant against waterborne microorganisms being present in medical in-house and dental unit water-lines (DUWL), considering a dilution effect of PAW when flushing the water bearing systems. The efficiency of PAW, activated under defined conditions (90 W for 30 min) in a lab unit, was studied towards eight different waterborne species focusing on 'Pseudomonas aeruginosa, Acinetobacter' spp. and 'Legionella' spp. present in water-lines. PAW, presenting low pH at < 3.2 and high oxidation-reduction potential (763 mV) and conductivity (963 μS/cm) values, was applied in defined units to determine the minimum volume amount added to water for bacterial reduction. Six species failed in growth when exposed to the double PAW volume unit after 30 min incubation. A fivefold volume excess provided sufficient activity to inactivate the waterborne microorganisms while only 'Acinetobacter baumannii' required a tenfold PAW surplus substantially reduction. Microorganisms show a species-specific susceptibility to PAW and cell count reduction strikingly correlates with added PAW volumes. PAW needs to be used in excess to achieve adequate cell reduction in aqueous environments, considering that dilution effects always accompany the disinfection. Our results indicate that PAW is a suitable disinfecting agent in a watery environment applicable for microbiological reduction in DUWLs.