Objetive: To evaluate and compare the antimicrobial efficacy of photodynamic therapy with and without ultrasonic activation of methylene blue against a multispecies biofilm developed in the root canals of human single-rooted teeth.

Methods: Eighty human single-rooted teeth were randomly allocated into four groups (n = 20): untreated multispecies biofilm (G1), conventional endodontic treatment (G2), photodynamic therapy (G3), and photodynamic therapy with ultrasonic activation of methylene blue (G4). The biofilm consisted of Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans, and Candida albicans and was cultured for 14 days. In G3, 0.005% methylene blue was used with a 5-minute pre-irradiation period, followed by irradiation with a 660-nm, 100-mW diode laser for 90 seconds. In G4, the photosensitizer was ultrasonically activated for 20 seconds before irradiation. Residual microbial load was quantified as colony-forming units per milliliter (CFU/ml). Overall differences were analyzed using the Kruskal-Wallis test, and pairwise comparisons were performed using the Mann-Whitney U test with Holm-Bonferroni adjustment. 

Results: Median microbial loads were 2.93 × 10⁶ CFU/ml in G1, 0 CFU/ml in G2, 1.65 × 10⁵ CFU/ml in G3, and 2.89 × 10⁴ CFU/ml in G4. Relative reduction compared with the untreated control group (G1) was 100.00% for G2, 94.37% for G3, and 99.01% for G4. Highly significant overall differences were observed among the groups (H [3] = 69.94; p < 0.001; ε² = 0.885). G4 showed a significantly lower microbial load than G3 (p < 0.001), with a large effect size (r = 0.80) and an estimated difference of 0.70 log units. 

Conclusion: Ultrasonic activation of methylene blue significantly enhanced the antimicrobial efficacy of photodynamic therapy against intracanal multispecies biofilm. Under the experimental conditions evaluated, this strategy may enhance PDT as an adjunctive procedure for endodontic disinfection.