The in vitro study compared the frequency of applications of ozonated propylene glycol using passive ultrasonic irrigation versus manual dynamic activation in the septic neutralization of root canals. Using 20 single-rooted teeth previously inoculated with Enterococcus faecalis. Biomechanical instrumentation was performed with the Protaper Next system, using ozonated propylene glycol as the irrigant through passive ultrasonic irrigation techniques and manual dynamic activation at different time points. The groups that received manual dynamic activation (MDA) showed CFU/ml: 5.03 × 105, 4.45 × 105, 4.00 × 105, 3.67 × 105, and 3.78 × 105, respectively, while with passive ultrasonic irrigation (PUI) the results were 4.45 × 105, 3.94 × 105, 3.55 × 105, 2.77 × 105, and 2.63 × 105 CFU/ml. It is determined that passive ultrasonic irrigation of the irrigant is the best way to enhance PGO3 penetration into the deeper regions of the root canal. By greatly increasing its antibacterial and disinfecting capacity, this method maximizes the efficacy of endodontic therapy and guarantees more thorough disinfection of difficult-to-reach places.