Background: Microbial contamination in dental unit waterlines (DUWLs) has recently become an important issue in the field of dental infection control. Using neutral electrolytic water as a new disinfecting method has attracted considerable attention. However, long term data about the effect of using neutral electrolytic water in clinical settings are scarce. This is the first study to evaluate the long term effectiveness of inhibition of bacterial proliferation using the purification system to supply neutral electrolytic water to refine the waterworks in DUWLs. Methods: Before the study, we investigated the actual levels of bacterial contamination in DUWLs. Then we did thorough cleaning of DUWLs and water samples were collected 6 dental units. Three dental units assigned as Group A had purification systems that used neutral electrolytic water, and the other 3 units were the Control group. Water samples were collected from the gargle water, high-speed handpieces and the three-way syringe. We utilized the equipment in Group A, and both groups were maintained for daily clinic work for 14 months. We counted the bacterial colony forming units (cfu) for each sample and identified the pathogenic bacterial species. Results: 3 and 14 months later, no microbes were detected during the study period in Group A whereas numbers of cfu which grew from the Control group increased and glucose non-fermenting gram-negative rod of possible pathogenic organisms to human were identified in the control groups. Conclusions: The water purification system using neutral electrolytic water was effective to control the proliferation of bacteria and could maintain a hygienic environment in DUWLs.