This study evaluated antimicrobial photodynamic therapy (aPDT) as an adjunct to endodontic treatment. Ten uniradicular teeth (control group (CG) = 4 (2 and test group (TG) = 6) with primary endodontic infections, from both genders, between 17 and 65 years old, were analyzed. Microbiological samples were collected before and after chemical-mechanical instrumentation (CMI), after aPDT (for the TG), and after the removal of the temporary restorations (second session). In TG, the aPDT was performed with 100 μg mL methylene blue and irradiated with low power laser (InGaAIP, 660 nm; 100 mW; 40 s) with a fiber-coupled optical laser. Another irradiation (3 J; 30 s; spot size of 3 mm ) was performed in the gingiva close to the apical foramen. The PCR was performed, after previous whole-genome amplification, for Enterococcus faecalis, Candida genus and Bacteria domain. For TG, a positive tooth for Candida spp. before of the CMI presented negative results in subsequent samples. Additionally, E. faecalis species was present in four samples before CMI, two after CMI, in one after the aPDT and was not detected at the second session. aPDT may be an effective adjunct therapy, resulting in a reduction (P = 0.0286) of the incidence of E. faecalis before root canal obturation.