Objective The persistence of pathogenic microorganisms in root canals is the most common reason for the failure of root canal treatment and the necessity of a root filling treatment, which results in an uncertain prognosis due to technical complexity and the variety of highly adaptable microorganisms. This study evaluated the effect of passive ultrasonic irrigation (PUI) on the outcome of the microbial analysis of root canal-treated teeth with persistent or recurrent apical inflammation in vivo. Materials and methods Sample collection was performed after root filling removal (sample S1, control group) and after PUI with NaCl (sample S2) using sterile paper points. In total, 19 samples were obtained. Quantification was performed by means of serial dilution of the samples. Subcultivated pure cultures were identified using MALDI-TOF MS complemented by the Vitek-2-System or PCR, followed by sequencing of the 16S rRNA gene. The results of the samples (S1 and S2) were evaluated regarding their bacterial count and composition. Results The total count of bacteria and the number of aerobic/facultative anaerobic microorganisms significantly increased in the S2-samples after application of PUI. The number of obligate anaerobic microorganisms showed an increase after PUI, although it was not significant. We detected 12 different aerobic/facultative anaerobic microorganisms before PUI, and in 21 cases after PUI. Two different obligate anaerobic microorganisms were found in S1 samples compared to nine different species in S2 samples. Conclusions PUI is a powerful method for detaching bacteria in infected root canals and enables a more precise analysis of the etiology of persistent endodontic infections. Clinical relevance This study indicates that PUI exerts a positive cleansing effect and adds to the accessibility of microorganisms during the application of bactericidal rinsing solution in root canal treatments.