Although bacterial contamination is widely researched in dental unit water systems, we have been unable to find any published reports to date about the presence and distribution of sulphate-reducing bacteria (SRB) in dental unit waterlines (DUWLs). The aim of this study was to evaluate microbial contamination and to determine the presence and distribution of SRB in DUWLs. One hundred twenty-three water samples were collected from the air-water syringes, high-speed drills and water sources from 41 dental units in Istanbul, Turkey. The counts of aerobic heterotrophic bacteria and SRB were investigated in the water samples. In addition, the presence of free-living amoebae (FLA) was examined. In this work, we evaluated microbial contamination and reported for the first time the distribution of SRB in DUWLs. We determined that only ten out of 123 water samples were able to meet the American Dental Association's limit (≤200 CFU ml( - 1)). SRB were observed in 102 out of 123 samples (82.9%). In addition, SRB were detected in all of the air-water syringes and high-speed drills. FLA were established in 103 out of 123 samples (83.7%).