The aim of the present study was to evaluate the efficacy of a between-patient disinfection procedures to maintain low bacterial counts in dental unit water line (DUWL) effluents, and control dental water line biofilms. Six dental units already in use, that had never been cleaned, were monitored for three weeks. During the first week only baseline contamination levels were assessed with no treatment of the system. In the second week lines were flushed with water for 30 s before treating each patient. During the third week, a disinfection procedure with 0.26% peracetic acid, followed by a water flush, was implemented before treating each patient. DUWL samples were collected both at the beginning and at the end of 216 dental procedures (72 during each period), plated on R2A agar and incubated at room temperature for seven days to obtain total bacterial counts in colony forming units per millilitre. To assess biofilm control, nine dental units (five never used and four old dental units with established biofilm) were used for 30 days in routine dental practice undergoing five between-patient DUWL disinfecting cycles every day. Water line samples were removed at baseline and at the end of the study and examined by scanning electron microscopy to determine the presence or absence of biofilms. A significant difference (P < 0.01) in mean DUWL bacterial counts was found between the three sets of observations. Biofilms were not present in any of the new dental units and a demonstrable reduction in the biofilms from the four dental units with previous presence of established biofilms was observed at the end of the study. In this study, a between-patient disinfection procedure consisting of flushing DUWL with peracetic acid with use of water was efficacious in the control of both microbial contamination of dental treatment water and dental water line biofilms.