Introduction: This in vivo study used molecular microbiology methods to evaluate the effects of passive ultrasonic irrigation (PUI) as a supplementary disinfecting step after root canal preparation. Methods: Samples were taken from 10 necrotic root canals of teeth with apical periodontitis before (S1) and after rotary nickel-titanium instrumentation using 2.5% NaOCl as the irrigant (S2) and then after PUI for NaOCl activation (S3). The parameters examined included the incidence of positive broad-range polymerase chain reaction (PCR) results for bacterial presence, the impact on bacterial diversity evaluated by PCR-denaturing gradient gel electrophoresis (DGGE), the quantitative bacterial reduction determined by real-time PCR, and the identification of persistent species by clone library analysis. Results: All S1 samples were positive for bacteria in all tests. Treatment procedures were significantly effective in reducing the incidence of positive results for bacteria, the number of bacterial cells (infectious bioburden), and the bacterial diversity (number of species and abundance). However, the supplementary PUI approach did not succeed in significantly enhancing disinfection beyond that achieved by chemomechanical preparation. Several bacterial species/phylotypes were identified in post-treatment samples that were positive for bacteria. Conclusions: Findings from this clinical study including a small sample size suggest that PUI can be ineffective in significantly improving disinfection of the main root canal after chemomechanical procedures. Copyright © 2013 American Association of Endodontists.