Objective: To analyze the antimicrobial activity of photodynamic therapy as an adjunct to conventional endodontic treatment, particularly against Enterococcus faecalis. Method and materials: A total of 42 single-rooted teeth obtained from 33 patients with apical periodontitis were included. Sampling was developed in three stages: (1) immediately after accessing the root canal, (2) after chemical and mechanical instrumentation, and finally, (3) after photodynamic therapy application. The bacterial load of each sample was quantified by seeding on blood agar plates and selective M-Enterococcus agar. All growing colonies were identified using MALDI-TOF (Bruker; matrix-assisted laser desorption/ionization time-of-flight), and the entire bacterial microbiota composition was determined in the first sample by PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis), using 16 rDNA primers and selective nucleotide sequencing. Results: The endodontic therapy obtained a mean reduction in the cultivable bacterial load of 1.12 log, whereas the photodynamic therapy combination significantly increased the bacterial clearance (P .0001). Viable cells of E faecalis were detected in 16.6% of root canals, with a mean value of 93 CFU per tooth, which was reduced to 67 and 9 CFU/tooth after conventional endodontic and photodynamic therapy treatments, respectively. Molecular E faecalis detection demonstrated that this species was present in 23.2% of baseline samples. DGGE analysis demonstrated the existence of a more complex microbiota than those observed using classical cultures. Conclusion: Photodynamic therapy as an adjunct to root canal treatment produces a significant reduction in E faecalis bacterial load, and it should be considered in the prevention of apical periodontitis. Keywords:  apical periodontitis, endodontics, Enterococcus, oral microbiota, photodynamic therapy