The aim of this study was to evaluate the bacterial contamination level and to determine the antibiotic susceptibility of the isolated bacteria from dental unit waterlines (DUWLs) in Istanbul. Bacterial quality of DUWLs is very important, as patients and dental staff are regularly exposed to water and aerosols generated by the unit. If opportunistic pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and Legionella pneumophila are present in DUWLs, patient and dental staff can be infected. One hundred water samples were collected from high-speed drills and input waters from 50 dental units. Aerobic heterotrophic bacteria counts and the presence of Legionella, Pseudomonas, oral streptococci, and Staphylococcus were investigated in dental unit waters and aerosol samples. In addition, the antibiotic susceptibility of the isolated and identified bacteria from DUWLs was examined. This research found that 37 out of 50 dental unit water samples exceeded the American Dental Association's limit of 200 colony-forming units (CFU)/mL(-1). Legionella, oral streptococci, and S. aureus were not detected in any water or aerosol samples, but P. aeruginosa was isolated in three DUWLs. Also, Pseudomonas and Staphylococcus were found in water and aerosol samples. Cefoperazone, ofloxacin, gentamicin, ciprofloxacin, and piperacillin were the most effective antibiotics against the isolated bacteria from DUWLs.