Water quality in the dental unit waterlines (DUWLs) is important to the patients and dental health care personnel as they are at risk of being infected with opportunistic pathogens such as Pseudomonas or Legionella species. In this study, a total of 86 samples were collected from DUWLs of 19 dental units in 11 Malaysian Armed Forces dental centres (MAFDC). 350 ml water sample was collected in sterile thiosulphite bags from the outlets of 3–way syringe, high speed handpiece, scaler, cup filler, independent water reservoir or the tap of the same surgery respectively. Samples were transported to the laboratory within 24 hours and kept in the refrigerator at 40C. 100ml of each sample was filtered through a 0.45 μm polycarbonate membrane filter. The filter was then inoculated onto plate count agar and incubated at 370 C for 24 hours, after which the formed colonies were enumerated. Another separate 100ml of water sample was poured onto buffered charcoal yeast extract agar and cetrimide agar to culture Legionnella and Pseudomonas respectively. Identification of these bacteria were confirmed by polymerase chain reaction and sequencing. Pseudomonas aeruginosa was detected in 9.5% of the samples but Legionnella was not detected in any of the samples. 77% of the samples met American Dental Association (ADA) recommendation of less than 200 cfu/ml. The result of this study showed that it is difficult if not impossible to eliminate biofilm from the DUWLs. Regular monitor of water quality from DUWL is required to maximise the health of the dental patients and dental health care personnel.