Objective: In order to evaluate the microbial contamination of different departments and explore the diversity of living bacterial community, the method of Propidium monoazid pretreatment combined with high-throughput sequencing technology was used to detect the living microbial community in the DUWLs (Dental unit waterlines) . Methods: A total of thirty-three dental chair units were selected from the Affiliated Hospital of Stomatology, Nanjing Medical University. Before patients arrived,Water samples were collected from each DCU using a three-in-one air/water syringe .After PMA pretreatment, the total DNA of living bacteria was extracted. After PCR amplification with bacterial universal primers, the macro genomic library was constructed for high-throughput sequencing and bioinformatics analysis.Results: In our study, results showed that when samples were pretreated with 1μL of PMA (final concebtraton:20μM) for 10 min and subsequently exposed to light irradiation for 5 min were optimal for detecting viable bacterial in samples of DUWLs. Results yielded that the output water of DUWLs was associatied with a high bacterial diversity and identified potential bacterial pathogenic sequence. The statistically different phyla between the two groups were Cyanobacteria, Actinobacteria, Chlamydiae, Aerophobetes, Chloroflexi, Bacteria_d_norank_k__unclassified, TM6 (P<0.05),The statistically different genera between the two groups were Legionella, Methyloversatilis, Obscuribacterales__ norank (P<0.05).Conclusions: The occurrence of potentially pathogenic microorganisms in these DUWLs demonstrated a potential infectious risk for both dental health care personnel and patients. Our study highlighted an ignored diversity of the DUWLs bacterial community in our country .Our findings also contribute a better appreciation of the potential infection risk associated with dental care and suggest the importance of better managing microbial quality in DUW. It is necessary to proceed to regular water quality control of DUWLs.