Water contamination in dental unit waterlines (DUWLs) is a potential source of healthcare-associated infection during dental care. The aim of this study was to evaluate the microbiological quality of DUWLs water from newly installed dental chairs in a French University Hospital. The microbiological quality of water from 24 new DUWLs initially disinfected by ICX Renew—prior to use of the dental units for patient treatment—was assessed for total culturable aerobic bacteria at 22°C and 36°C, Legionella sp. , Pseudomonas aeruginosa , and total coliforms. Among the 24 samples analyzed, 21 were compliant with the water quality levels: 19 had no bacteria, and 2 contained only 4 and 1 CFU/mL for total culturable aerobic bacteria at 22°C and 36°C, respectively. Three samples were non-compliant due to contamination by P. aeruginosa (4, 2, and 2 CFU/100 mL). Controlling and preventing the microbiological contamination of DUWLs, especially by pathogenic bacteria, at the time of the installation of the new dental chairs are crucial to prevent healthcare-associated infection in dentistry. IMPORTANCE Dental unit waterlines (DUWLs) of new dental chairs may be contaminated before their first clinical use, so an initial shock disinfection is crucial at the time of their installation. The microbiological analyses are crucial to control the water quality of DUWLs before their first clinical use because their disinfection does not guarantee the elimination of all bacteria.