AimThis in-vitro investigation aimed to assess the antibacterial effectiveness of photo-sonodynamic treatment using methylene blue (MTB)-incorporated poly(D, L-Lactide-Co-Glycolide) acid (PLGA)-nanoparticles for the disinfection of root canals. MethodsThe synthesis of PLGA nanoparticles was achieved using a solvent displacement technique. The morphological and spectral characterization of the formulated PLGA nanoparticles were carried out using scanning electron microscopy (SEM) and Transformed-Fourier infrared spectroscopy (TFIR), respectively. One hundred human premolar teeth were sterilized and then their root canals were infected with Enterococcus faecalis (E. faecalis). Later, the bacterial viability evaluation of the following 5 research groups was conducted: (a) G-1: specimens treated with a diode laser; (b) G-2: specimens treated with antimicrobial photodynamic therapy (aPDT) and 50 µg/mL of MTB-incorporated PLGA nanoparticles; (c) G-3: specimens treated with ultrasound (US); (d) G-4: specimens treated with US and 50 µg/mL of MTB-incorporated PLGA nanoparticles; and (e) G-5: control group consisting of specimens that did not undergo any treatment. ResultsUnder SEM, the nanoparticles exhibited a uniform spherical shape and were around 100 nm. The formulated nanoparticles’ size was validated through zeta potential analysis utilizing dynamic light scattering (DLS). The TFIR images of both PLGA nanoparticles and MTB-incorporated PLGA nanoparticles exhibited absorption bands ranging from around 1000 to 1200/cm and nearly from 1500 to 1750/cm. The G-5 samples (control group) demonstrated the greatest viability against E. faecalis, followed by G-3 (US-conditions specimens), G-1 (diode laser-conditioned specimens), G-2 (aPDT + MTB-incorporated PLGA-nanoparticles-conditioned specimens), and G-5 (US + MTB-incorporated PLGA-nanoparticles-conditioned specimens). Significant statistical differences (p < 0.05) were observed among all research groups, including both the experimental groups and control group. ConclusionThe combination of US via MTB-incorporated PLGA nanoparticles exhibited the most effective eradication of E. faecalis, suggestive of a promising therapeutic modality against E. faecalis for disinfecting root canals with complex and challenging anatomy.