Background Apical periodontitis is caused by bacteria present in the root canal space. The removal of the infection is crucial to obtain healing. Canal irrigation is among one of the most important steps in eliminating bacteria. Sodium hypochlorite (NaOCl) is still the preferred irrigant due to its disinfecting and pulpal dissolution abilities. Heating NaOCl improves those abilities. However, the ability of intracanal heated NaOCl to kill bacteria has not yet been evaluated. Objectives This study compared the disinfecting ability of different irrigation regimens using NaOCl with and without sonic and ultrasonic agitation, and with and without intracanal heating of NaOCl. Methods The canals of extracted mandibular premolars were prepared, sterilized and infected with E. faecalis for 28 days. The canals were then assigned to eight groups of 10 teeth depending on the NaOCl irrigation protocol. Group CONV: conventional irrigation with syringe and needle; Group END: NaOCl sonic agitation with EndoActivator; Group EDD: NaOCl sonic agitation with EDDY; Group PUI: NaOCl passive ultrasonic agitation; Group H: intracanal heating of NaOCl; Groups END-H, EDD-H and PUI-H: NaOCl agitation with EndoActivator, EDDY and passive ultrasound, respectively, followed by intracanal heating of NaOCl. The canals were sampled before (S1) and after (S2) the different irrigation protocols were performed, the colony-forming units were counted and the percentage of bacteria reduction was calculated for each group.