The purpose of this study was to compare the in vitro intracanal bacterial reduction produced by using two instrumentation techniques and different irrigation methods. Root canals inoculated with Enterococcus faecalis were prepared by using the following techniques and irrigants: alternated rotary motions (ARM) technique, hand nickel-titanium files and 2.5% sodium hypochlorite (NaOCl) as irrigant; ARM technique and combined irrigation with 2.5% NaOCl and citric acid; ARM technique and combined irrigation with 2.5% NaOCl and 2% chlorhexidine gluconate; and Greater Taper rotary files, using 2.5% NaOCl as irrigant. Controls were instrumented by using the ARM technique and irrigated with sterile saline. Canals were sampled before and after preparation. After serial dilution, samples were plated onto Mitis-Salivarius agar, and the colony forming units that were grown were counted. All test techniques and solutions significantly reduced the number of bacterial cells within the root canal (p < 0.05). There was no significant difference between the experimental groups (p > 0.05). Nonetheless, all of them were significantly more effective than the control group (p < 0.05). These findings support the importance of using antimicrobial irrigants during the chemomechanical preparation, regardless of the solutions or instrumentation techniques used.