Introduction: The aim of this study was to evaluate the in vitro antibacterial activity by atmospheric pressure nonequilibrium plasmas (APNPs) as an effective approach against bacterial biofilms in root canal systems during endodontic therapy. Methods: Sterile cover slips were placed into the wells of tissue culture plates to permit the formation of Enterococcus faecalis biofilms. Biofilms were treated for 5 minutes with APNPs or 2% chlorhexidine digluconate (CHX). The viability of biofilm bacteria was analyzed by staining and confocal laser scanning microscopy. In addition, infected single-rooted teeth were exposed to APNPs or 2% CHX for 5, 10, and 15 minutes. After treatment, the root canals were flushed, and the resulting suspensions were inoculated onto brain-heart infusion agar to assess bacterial survival. Finally, micro computed tomography scanning was used to observe and verify the root canal systems relative to the antibacterial effects obtained. Results: Treatment for 5 minutes with APNPs or 2% CHX killed the majority of bacteria in the E. faecalis biofilms. Moreover, APNP treatment was as effective as 2% CHX for inactivating bacteria in infected root canals (P > .05). Bacterial survival after treatment with APNPs or 2% CHX remarkably reduced with increasing exposure times (P < .05). There was no significant difference between bacterial survival in complex root canal systems and simple straight canals (P > .05). Conclusions: APNPs can be an effective adjunct to standard endodontic antimicrobial treatment. (J Endod 2012;38:545-549)