This study aimed to clinically evaluate the disinfection efficacy of chlorine dioxide (ClO2) for used dental instruments. An imprint culture technique demonstrated that ultrasonic cleaning of intraorally applied dental mirrors in 0.02% ClO2 for 10 minutes resulted in compete removal of microorganisms for 10 subjects. Hepatitis C virus (HCV) RNA was detected by real-time polymerase chain reaction on periodontal curettes after subgingival scaling in four HCV-infected patients and was completely removed by the same treatment procedure. Therefore, the combination of ultrasonic cleaning with ClO2 may provide an alternative to toxic disinfectants, such as glutaraldehyde and sodium hypochlorite, for disinfecting dental instruments.