<ul><li><strong>BACKGROUND</strong>: Dental unit waterlines (DUWLs) are ideal nvironment for development of microbial biofilms. Microbial contamination of water in DUWLs is thought to be the result of biofilm formation as it could serves as a haven for pathogens. The aim of this study was to assess microbial quality of water in dental unit waterlines of dental units located at the dental school of Isfahan University of Medical Sciences.</li><li><strong>METHODS</strong>: Water samples were collected from air/water syringe and high-speed handpiece. Generally, 100-200 ml water samples were collected aseptically in sterile containers with sodium thiosulfate at the beginning of the day after a 2 minute purge. Samples were transferred to the laboratory in insulated box with cooling packs and examined for total viable heterotrophic bacteria and fungi.</li><li><strong>RESULTS</strong>: The heterotrophic plate count levels were significantly exceeded the American Dental Association recommendations for DUWL water quality (&lt; 200 CFU/ml), in both air/water syringe (84%, CFU/ml: 500-20000) and highspeed handpiece (96%, CFU/ml: 710-36800) samples. However, there was no significant difference between the level of contamination in the air/water syringe and high-speed handpiece. Fungi were found in 28% and 36% of air/water syringe and high-speed handpiece samples, respectively; and filamentous fungi were the most frequently isolated fungi.</li><li><strong>CONCLUSIONS</strong>: DUWLs should be subjected to routine microbial monitoring and to a decontamination protocol in order to minimize the risk of exposure to potential pathogens from dental units.</li><li><strong>KEYWORDS</strong>: Biofilm, Dental Unit Waterlines, Microbial Quality, Water.</li></ul>