Aim: The aim was to evaluate the bacterial reduction achieved by ProTaper Next (PTN) system in root canals contaminated with Enterococcus faecalis. ProTaper Universal (PTU) and WaveOne (WO) systems were used as references for comparison. Materials and methods: A hundred human mandibular premolars were selected and ninety-five were contaminated with E. faecalis for 4 weeks. Then the teeth were randomly divided into six groups (n = 15): PTN + 2.5% NaOCl; PTU + 2.5% NaOCl; WO + 2.5% NaOCl; PTN + saline solution; PTU + saline solution; and WO + saline solution. Positive and negative control groups were created with five specimens each. The irrigation volume was standardised for all experimental groups. Samples taken before (S1) and after (S2) chemomechanical procedures were cultured and the colony-forming units (CFUs) were counted. The paired t-test was used for intragroup analysis, and one-way ANOVA for intergroup analysis. When significant differences were found amongst the groups, the Tukey test was used. The significance levels were set at 5% (P 0.05). Results: After root canal instrumentation (S2), there was a significant reduction in bacterial load in all groups (P 0.01). The WO/saline solution group showed a lower level of bacterial reduction when compared with the other tested groups (P 0.01). Groups irrigated with NaOCl showed a higher level of bacterial reduction when compared with saline solution irrigation (P 0.01), with no statistical differences amongst the systems used (P > 0.05). Conclusion: The PTN system was as effective as PTU and WO in the removal of bacteria during root canal treatment. Keywords: Enterococcus faecalis, ProTaper Next, Root canal instrumentation, sodium hypochlorite