BackgroundSecondary/persistent endodontic infection can be the outcome of failure of endodontic treatment. Photo-activated disinfection (PAD) can be a useful adjunct to mechanical and antimicrobial agents in eliminating endopathogenic microorganisms. In this study, we evaluated the effect of PAD on diversity and count of microbiota related to secondary/persistent endodontic infections. Materials and methodsRoot canal samples were taken using sterile paper points from the root canals of 14 patients with secondary/persistent endodontic infections after removing the root-canal filling materials. PAD was performed on teeth with toluidine blue O (TBO) in combination with diode laser. Then re-sampling was conducted from the canal root using sterile paper points and transferred to transport medium. The samples were plated and pure cultures of the target microorganisms were then isolated and identified by analytical profile index (API ® 20A) assays and 16S ribosomal RNA gene sequencing. ResultsBefore TBO-PAD, a total of 31 cultivable isolates could be retrieved; 25.8% of the isolated species were obligate anaerobic or microphilics including Veillonella parvula, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Propionibacterium acnes, and Lactobacillus acidophilus, and 74.2% of the isolated species were facultative anaerobic such as Enterococcus faecalis, Actinomyces naeslundii, L. rhamnosus, L. casei, Streptococcus sanguinis, S. mitis, and Candida albicans. According to this in vivo study, the diversity and count of microbiota in root canal-treated teeth were decreased after TBO-PAD, so that E. faecalis, V. parvula, and C. albicans were the microorganisms that recovered after PAD. ConclusionTBO-PAD is an effective approach that exhibited anti-microbial potential activity against microbiota involved in secondary/persistent endodontic infection.