Objectives: Although dental unit waterlines (DUWLs) could harbor biofilms that pose an infection risk to patients and staff, data on infection control factors related to the microbial contamination of DUWLs in Korea remains limited. Therefore, in this study, we aimed to analyze the microbial load in DUWL according to infection control factors using a survey. Methods: In this cross-sectional study, we surveyed 58 dental institutions for characteristics (chair number, daily patient load, and accreditation status) and infection-control status (written guidelines, designated managers, monitoring, and staff training). Simultaneously, we examined the microbial contamination levels of the high-speed handpieces and three-way air-water syringes via water sampling, expressing contamination as arithmetic and geometric means (GM)±geometric standard deviation (GSD). We used the Mann-Whitney U test to compare bacterial contamination according to institutional characteristics and infection control factors (p<0.05). Results: Overall GM contamination was 1,141 and 411 CFU/mL for high-speed handpieces and syringes, respectively, exceeding the CDC guideline of ≤500 CFU/mL. We observed significant differences in microbial loads according to the institution type, unit chair count, and average patient count. Moreover, the existence of infection control guidelines revealed significant effects. Conclusions: DUWL microbial quality varied according to the clinic size and infection control program quality. Institutions with detailed guidelines, routine surveillance, and skill-based training have achieved better microbial control. Standardized guidelines and incentivized training could help reduce infection control gaps, especially in small private clinics.