Objective：In order to evaluate the microbial contamination of different departments and explore the diversity of living bacterial community，the method of propidium monoazid（PMA） pretreatment combined with high-throughput sequencing technology was used to detect the living microbial community in the dental unit waterline（DUWL）. Methods：A total of 33 dental chair units（DCU）were selected from the Affiliated Stomatological Hospital of Nanjing Medical University，with 23 from endodontics department（group E）and 10 from periodontics department（group P）. 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 macrogenomic library was constructed for high-throughput sequencing and bioinformatics analysis. Results：Samples were pretreated with 1 μL of PMA（20 μmol/L）for 10 min and subsequently exposed to light irradiation for 5 min，which were optimal for detecting viable bacterial in samples of DUWL. The output water of DUWL 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）. Conclusion：The occurrence of potentially pathogenic microorganisms in these DUWLs demonstrated a potential infectious risk for both dental health care personnel and patients. Attention should be paid to the diversity of DUWL bacterial communities . It is necessary to proceed to regular water quality control of DUWLs.