Objective: To compare the antimicrobial effectiveness of passive ultrasonic irrigation (PUI) and continuous ultrasonic irrigation (CUI) against a multispecies biofilm by counting colony-forming units per milliliter (CFU/mL).

Methods: An in vitro experimental study was conducted using 60 human single-rooted premolars. Two experimental groups were established: PUI (n = 20) and CUI (n = 20), along with 10 positive controls and 10 negative controls. The multispecies biofilm consisted of Enterococcus faecalis, Candida albicans, and Staphylococcus aureus. Following chemomechanical preparation with reciprocating instrumentation and 5.25% sodium hypochlorite, the corresponding ultrasonic activation protocols were applied. Microbial load was assessed at three time points: baseline, after chemomechanical preparation, and after ultrasonic activation. Nonparametric statistical tests were used for within-group and between-group comparisons.

Results: Both groups showed a statistically significant reduction in microbial load across the assessed stages (p < 0.001). In the PUI group, the median decreased from 1.24 × 10⁷ CFU/mL at baseline to 4.90 × 10² CFU/mL after activation. In the CUI group, it decreased from 7.20 × 10⁶ CFU/mL to 2.55 × 10¹ CFU/mL. The between-group comparison after activation showed a statistically significant difference (p < 0.001; r = 0.61), with a lower residual microbial load in the CUI group. Counts below 100 CFU/mL were found in 85% of CUI specimens, compared with 25% of PUI specimens.

Conclusions: Both techniques significantly reduced the microbial load of the multispecies biofilm. Under the experimental conditions of this study, CUI resulted in a lower residual microbial load than PUI; however, these findings do not directly establish clinical superiority.