The study aimed to evaluate the antimicrobial effect of photodynamic therapy (PDT) with the use of Toluidine Blue (TB) on extracted teeth infected with biofilms of Enterococcus faecalis. Fifty-four extracted teeth with single-roots and single canals were mechanically shaped, autoclaved, and contaminated with E. faecalis. They were randomly divided into six groups: two groups were negative and positive control groups, two groups were subjected to mechanical instrumentation and PDT with different pre-irradiation times and irradiation times, and two groups were subjected to chemo-mechanical endodontic treatment and PDT with different pre-irradiation times and irradiation times. In PDT groups, after the application of TB, the canals were irradiated with a diode laser of wavelength 635 nm, with a fiber diameter of 200 μm and 100 mW of power in continuous mode. The bacterial load was evaluated using a BioTimer Assay protocol. The greatest reduction of bacterial load was observed in groups of combined PDT with chemo-mechanical treatment. The reductions of bacterial load in groups of combined PDT with chemo-mechanical treatment, and in the positive control group, were significant (p < 0.01) when compared to that of the negative control group. Photodynamic therapy as an adjunctive modality may improve the disinfection capacity of conventional endodontic treatment against E. faecalis.