Aim: The aim of this study was to evaluate the microbial contamination of dental unit waterlines (DUWLs) and characterize the microbial communities of biofilms in dental chair units (DCUs) from different specialties in Ukrainian dental clinics. Materials and Methods: A multicentre study was performed between January 1, 2020, to December 31, 2022. Dental water samples and biofilm samples were obtained from 191 DCUs at eighteen dental clinics from seven regions of Ukraine. The genomic DNA of the biofilm samples was extracted, then 16S rDNA were amplified and sequenced. Results: A total of 1,146 dental water samples were collected, of which 57,4% samples did not meet microbiological parameters of Ukrainian National Standard on drinking water. Sequencing results showed significant differences in bacterial community structure between dental specialties. The largest specific weight of biofilm samples with high bacterial concentrations were detected from orthodontics (54.2%), prosthodontic (47.5%), and oral surgery (44,3%). The 16S rDNA gene sequencing showed high diversity of bacteria (311 genera) were detected in the biofilm samples. Amount of potential human pathogens were detected in the biofilm samples, including Pseudomonas aeruginosa (33.7%), Escherichia coli (27.3%), Enterococcus faecalis (17.4%), Enterococcus faecium (9.5%), Serratia marcescens (6.8%), Stenotrophomonas maltophilia (5.9%), Staphylococcus aureus (5.1%), Burkholderia cepacia (4.3%), Acinetobacter lwoffii (4.8%), Enterobacter cloacae (4.6%), Klebsiella oxytoca (4.2%), Streptococcus pneumoniae (3.9%), Streptococcus pyogenes (2.6%), and Streptococcus sp, (1.9%). Conclusions: The most water quality of the DUWLs tested failed to reach the Ukrainian drinking water standard. Furthermore, most DCUs contained pathogens which poses a risk of infection for patients.