ObjectiveTo compare the effect of different ways of water supply and whether or not drain water from waterlines for overnight on bacterial counts in dental unit waterlines(DUWLs). MethodsIn the first phase, 6 sets of DUWLs were randomly divided into 2 groups (external storage tank water supply group and municipal water supply group); in the second phase, 6 sets of DUWLs were all changed to use external storage tank and randomly divided into 2 groups (draining water for overnight group and without draining water for overnight group), bacterial count before and within one week of disinfection  between two groups at two phases were compared respectively. ResultsIn the first phase, handpiece water of DUWLs was most seriously contaminated by bacteria, the average colony count was 4 117 CFU/mL, qualified rate was 15.38%. Before disinfection, no significant difference in bacterial count were found among each groups (all P>0.05), bacterial count of DUWLs of all groups severely exceeded the standard(all >3 000 CFU/mL). Comparison of bacterial count in DUWLs from different water supply routes after disinfection was not significantly different on day 1(P>0.05), but were significantly different at day 27(all P<0.05). On the second day after disinfection, municipal water supply group began to exceed bacteria standard; on the third day after disinfection, external storage tank group began to exceed bacteria standard. Bacterial count in DUWLs after disinfection between draining water for overnight group and without draining water for overnight group was no significantly different on day 1(P>0.05) , but were significantly different on day 2-7(all P<0.05). On the fourth day of disinfection, bacterial count of without draining water for overnight group exceeded standard. On day 7 of disinfection, bacterial count in draining water for overnight group exceeded＞100 CFU/mL. ConclusionUse of external storage tank, daily change of sterile distilled water, and daily emptying water for overnight can effectively reduce bacterial count in DUWLs.