Objective <p>This study evaluated the effect of different power settings in ultrasonic activation, combined with antimicrobial and non-antimicrobial irrigants, on Enterococcus faecalis biofilm reduction and cell viability.</p> Materials and methods <p>A total of 150 root canals of human uniradicular teeth were prepared to size #50.05 and inoculated with E. faecalis for 21 days. Specimens were assigned to groups according to ultrasonic activation protocols (activation at lower or intermediate power and no activation) and irrigant (2.5% NaOCl or phosphate-buffered saline) used in ultrasonically activated irrigation. Biofilm reduction was assessed by colony-forming unit (CFU) counts and data were analyzed using two-way ANOVA and Tukey’s post hoc test (<i>p</i> &lt; 0.05). Biofilm structure and bacterial cell viability were qualitatively evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM).</p> Results <p>Significant differences in CFU counts were observed between irrigants, ultrasonic power settings, and their interaction (<i>p</i> &lt; 0.001). NaOCl 2.5% promoted greater microbial reduction regardless of the activation protocol (<i>p</i> &lt; 0.05). Intermediate power resulted in the lowest CFU counts (<i>p</i> &lt; 0.05), and showed greater qualitative biofilm disruption and reduced cell viability with both irrigants, particularly in the middle and apical thirds.</p> Conclusions <p>Intermediate ultrasonic power improves root canal cleaning by enhancing biofilm disruption and reducing bacterial cell viability, even without antimicrobial irrigants. Precise adjustment of ultrasonic power should be regarded as a critical parameter to maximize cleaning and disinfection efficacy.</p> Clinical Relevance <p>Adjusting ultrasonic power to an intermediate level improves biofilm removal and reduces bacterial viability, enhancing root canal cleaning even when non-antimicrobial irrigants are used.</p>