Objective To understand the current status and influencing factors of microbial contamination of dental unit waterlines (DUWLs) of secondary and lower medical institutions in Wuhan, and provide scientific basis for formulating prevention and control measures. Methods A stratified convenience sampling method was adopted to conduct questionnaire survey on 248 dental medical institutions in 15 districts of Wuhan City, DUWLs from 44 medical institutions were sampled and detected according to hospital levels. Results Among disinfection and maintenance management measures for source water and waterlines, the highest implementation rate was the installation rate of anti-suction dental handpiece (73.39%, 182/248), but only 16.48% (30/182) of institutions regularly conducted the detection on anti-suction function; The lowest was the daily disinfection rate of water storage tanks (17.53%, 17/97). A total of 132 water specimens were collected from 44 medical institutions, with a qualified rate of microbial detection of 56.06%. The qualified rates for secondary, primary, and unclassified medical institutions were 77.78%, 50.00%, and 50.72%, respectively, with statistically significant differences (P<0.05). The qualified rates of microbial detection for source water, handpiece water, and three-way syringe water were 59.09%, 50.00%, and 59.09%, respectively, with no statistically significant difference (P>0.05). Univariate analysis results showed that medical institutions that regularly conducted chemical disinfection on DUWLs, understood DUWLs waterline cleaning and disinfection standards, regularly performed microbial monitoring on diagnosis and treatment water, qualified source water, and flushed waterlines for 3 minutes before and after daily consultations had a higher qualified rate of microbial detection in DUWLs diagnosis and treatment water, and the differences were all statistically significant (all P<0.05). Conclusion The contamination status of DUWLs in secondary and lower medical institutions in Wuhan is relatively serious. It is recommended to focus on strengthening the training of relevant regulations, installing inlet filters, regularly detecting the anti-suction function of dental handpieces, implementing chemical disinfection and microbial monitoring on waterlines, standardizing the implementation of flushing before and after diagnosis and treatment, and strengthening the disinfection and use management of independent water storage tanks to control contamination.