BMC Oral Health. 2025 Jul 5;25(1):1113. doi: 10.1186/s12903-025-06493-2.ABSTRACTOBJECTIVES: To evaluate the antibacterial effectiveness of stepwise intraoperative activation of the root canal irrigant, both alone and when combined with conventional ultrasonic activation, compared to the traditional syringe irrigation method in oval root canals.METHODS: Sixty single-rooted maxillary second premolars were selected according to specific criteria. After creating an access opening and root canal patency in all samples, a cycle of autoclave sterilization was conducted. Enterococcus faecalis suspension was inoculated into the root canals and incubated at 37 °C for 21 days. After bacterial contamination, the specimens were assigned to four experimental groups (n = 10 per group): conventional syringe irrigation (CSI); stepwise intraoperative activation (SIA), consisting of the ultrasonic activation of 2 mL of 5.25% NaOCl solution for 20 s following irrigation between instrumentation files; conventional ultrasonic irrigation (CUI), consisting of the passive intermittent ultrasonic activation of 2 mL of 5.25% NaOCl in three successive cycles of 20 s each at the end of the mechanical instrumentation; and stepwise intraoperative activation plus conventional ultrasonic irrigation (SIA + CUI), consisting of a combination of SIA and CUI; in addition to positive control; and negative control groups (n = 10). At the end of the chemo-mechanical preparation, a bacterial sampling was conducted to determine the number of colony-forming units per mL (CFU//mL), and the outcomes were compared with one-way ANOVA and Games-Howell post hoc test with the significance level set at 5%.RESULTS: None of the tested irrigation protocols were able to fully eliminate the intracanal bacterial infection. However, all protocols succeeded in reducing the bacterial burden within the oval root canals considerably (p