This study assessed the intratubular antibacterial ability of different activated irrigations after chemical mechanical preparation. Seventy-two palatal root canals of upper molars were infected with Enterococcus faecalis for 4 weeks, and then initial bacterial collection from the main root canal was performed. The root canals were prepared by using a WaveOne Gold large (45/.05) and distributed into 6 groups according to the activation of the final irrigation: ultrasonic activation (UA), XP-Endo Finisher (25/.00), XP Clean (25/.02), EasyClean (25/.04) in reciprocating motion and continuous rotary motion (ECRot), and conventional irrigation. After final irrigation, another bacterial collection from the main root canal was performed, and the root was sectioned transversely in three-thirds and stained for analysis by confocal laser microscopy. Intratubular bacteria were collected through dentin powder and plated for bacterial viability analysis. Intergroup and intragroup comparisons were performed by using analysis of variance and repeated measures analysis of variance, respectively, both at 5% significance. ECRot had higher antibacterial ability than UA (p<0.05), and both were superior to the other groups (p<0.05) in both methodologies. It can be concluded that activation of final irrigation enhances the disinfection of the root canal system, and activators have different efficacies