Introduction This in vitro study aimed to investigate the antibacterial effects of photodynamic therapy (PDT) with methylene blue (MB) or toluidine blue (TB) (both at 15 μg/mL) as a supplement to instrumentation/irrigation of root canals experimentally contaminated with Enterococcus faecalis. Methods Seventy extracted teeth had their root canals contaminated with an endodontic strain of E. faecalis for 7 days, instrumented with nickel-titanium instruments and irrigated either with 2.5% NaOCl or with 0.85% NaCl, and then randomly distributed into four experimental groups: MB/NaOCl (PDT with MB and NaOCl as the irrigant), TB/NaOCl (PDT with TB and NaOCl as the irrigant), MB/NaCl (PDT with MB and NaCl as the irrigant), and TB/NaCl (PDT with TB and NaCl as the irrigant). For PDT, the photosensitizer remained in the canal for 2 minutes before exposed to red light emitted from a diode laser for 4 minutes. Samples were taken before and after instrumentation/irrigation and following the specific PDT procedure for each group, plated onto Mitis-salivarius agar and the colony forming units counted. Results Regardless of the irrigant used (NaOCl or NaCl), instrumentation significantly reduced bacterial counts in comparison to the baseline (p < 0.001). NaOCl as the irrigant was significantly more effective than NaCl, and this difference persisted after PDT, irrespective of the photosensitizer used (p < 0.05). PDT with either MB or TB did not significantly enhance disinfection after chemomechanical preparation using NaOCl as irrigant (p > 0.05). No significant differences were observed between the two photosensitizers (p > 0.05). Conclusion These in vitro results suggest that PDT with either MB or TB may not exert a significant supplemental effect to instrumentation/irrigation procedures with regard to intracanal disinfection. Further adjustments in the PDT protocol may be required to enhance predictability in bacterial elimination before clinical use is recommended.