Objective: To evaluate the efficacy of two sonic-powered irrigation systems, EDDY and EndoActivator, in killing intracanal biofilms. Methods: Eighty-three instrumented, autoclaved single-rooted human premolars were inoculated with Enterococcus faecalis (ATCC-29212) for 21 days to generate mature, single-species biofilms. The teeth were devided into four groups: irrigation with saline (N = 11); irrigation with 2% NaOCl without agitation (N = 24); irrigation with 2% NaOCl with agitation by the EndoActivaor (N = 24); irrigation with 2% NaOCl with agitation by the EDDY (N = 24). Colony-forming unit (CFU) counts and XTT assay were used to evaluate bacterial load. Additional teeth were split into root-halves for fluorescence staining to examine the percentages of live/dead bacteria in the dentinal tubules present in different canal locations (coronal, mid-root and apical portions). Results: Agitation of 2% NaOCl by EDDY or EndoActivator reduced bacteria load more proficiently than the use of 2%NaOCl without agitation (p < 0.05). No significant difference was detected between the two sonic-powered irrigant agitation systems (p > 0.05). Confocal laser scanning microscopy indicated that both devices had better intratubular bacteria killing efficacy than the use of 2% NaOCl only in all parts of the canal space (p < 0.05), with no significant difference detected between EDDY and EndoActivator (p > 0.05). Elimination of intracanal and intratubular bacteria were less efficient in the apical portion of the canal space for all experimental groups. Conclusion: The two sonic-powered irrigant activation systems demonstrated comparable intracanal bacteria reduction efficacy when 2% NaOCl was used for disinfecting E. faecalis biofilms in single-rooted teeth. Clinical significance: With the use of 2% NaOCl as irrigant, the EDDY and EndoActivator sonic-powered irrigant agitation systems have improved and equivalent intracanal bacteria reduction efficacy but are incapable of completely killing all bacteria that resided within the dentinal tubules of root canals.