Legionella pneumophila and Pseudomonas aeruginosa are common colonizers of water environments, particularly dental unit waterlines. The aim of this study was to assess whether the technical, functional and structural characteristics of dental units can influence the presence and the levels of opportunistic pathogens. Overall, 42 water samples were collected from dental units in a teaching hospital in Palermo, Italy, including 21 samples from the 21 taps supplied by the municipal water distribution system and 21 samples from oral rinsing cups at 21 dental units. L. pneumophila was present in 16 out of 21 water samples (76.2%) from dental units, and the median concentration was higher in samples from oral rinsing cups than in those from taps (P < 0.001). P. aeruginosa was equally distributed in water samples collected from oral rinsing cups and from taps. Some characteristics of dental units (age, number of chairs per room, number of patients per day and water temperature) were slightly associated with the presence of P. aeruginosa, but not with contamination by L. pneumophila. Our experience suggests that L. pneumophila is frequently detected in dental units, as reported in previous studies, whereas P. aeruginosa is not a frequent contaminant. As a consequence, microbiological control of water quality should be routinely performed, and should include the detection of opportunistic pathogens when bacterial contamination is expected.