This study aimed to evaluate the antibacterial effect of three final irrigation protocols and to compare their ability to remove the smear layer and debris from the root canal. Methods: Sixty-three single-rooted human teeth were inoculated with Enterococcus faecalis for 14 days. The teeth were divided into a positive control group (N = 3) and three treatment groups (N = 20) as follows: final irrigation with saline solution (control group), irrigation with 5.25% NaOCl ultrasonically activated with EndoUltra (EU), standard needle irrigation with Qmix 2in1 (Qx) and irrigation with 5.25% NaOCl activated using SiroLaser Blue (SB). The bacterial load was evaluated by analyzing the colony-forming units (CFU/mL). Selected specimens were split longitudinally and examined using scanning electron microscopy in order to determine the presence of a smear layer and debris. Statistical analyses were performed using one-way ANOVA and the Kruskal−Wallis rank-sum test. Results: Activation of NaOCl with EndoUltra or SiroLaser Blue was superior at reducing intracanal bacteria relative to standard needle irrigation with Qmix 2in1 solution (p < 0.05). Even though SiroLaser Blue showed the best results for removing the smear layer and debris, no significant differences were detected between the groups (p > 0.05). Conclusions: Final irrigation with 5.25% NaOCl ultrasonically activated using EndoUltra or SiroLaser Blue demonstrated a higher efficacy in bacterial reduction than standard needle irrigation with Qmix 2in1.