Bacterial contamination of dental unit waterlines (DUWLs) was evaluated by using ATP-bioluminescence analysis and conventional culture method. Water samples (n=44) from DUWLs were investigated for heterotrophic bacteria by culture on R2A agar, which ranged from 1.4×103 to 2.7×105 CFU/mL. The ATP-bioluminescence results for DUWL samples were ranged from 6 to 1189 RLU and obtained within one minutes. These results were well correlated with the culture results (r=0.727-0.855). We conclude that differences in the bacterial contamination of each water supply were confirmed by the ATP-bioluminescence assay. This method would be potentially useful for rapid and simple monitoring of DUWL bacterial contamination