Isolation of teeth with rubber dams is an important procedure for infection control in dentistry, especially in endodontic treatment. This study surveyed the prevalence of rubber dam usage in nonsurgical root canal treatment (RCT) by dentists under the National Health Insurance system in Taiwan. Methods: A total of 1,332 completed RCT cases were randomly selected from a large database from the Bureau of National Health Insurance in Taiwan in 2004. The radiographs and dental charts of the selected cases were evaluated for the prevalence of rubber dam usage in RCT. The frequencies of rubber dam usage for RCT by dentists were compared between hospitals and private dental clinics and among six different regions in Taiwan. Results: The overall prevalence of rubber dam usage for RCT by dentists under the National Health Insurance system in Taiwan was 16.5%. The frequency of rubber dam usage for RCT by dentists in hospitals (32.8%) was significantly higher than that (10.3%) in private dental clinics (p < 0.0001). However, there was no significant difference in the frequency of rubber dam usage for RCT by dentists among six different geographic regions in Taiwan. Conclusion: The prevalence of rubber dam usage for RCT by dentists in Taiwan is relatively low. Because rubber dam isolation of an endodontically-treated tooth can provide better infection control, increase patient protection, and improve treatment efficiency, there is an urgent need to advise dentists in Taiwan to use rubber dams for every RCT case.