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Aim:The aim of this study was to compare the bacterial reduction achieved with reciprocating instruments such as Reciproc® R25 (VDW GMBH, Munich, Germany) and Reciproc® R25 combined with rotary file Mtwo 40.04 (VDW GMBH) during root canal preparation.Materials and Methods:Fifty mesiobuccal root canals of maxillary molars were contaminated with Enterococcus faecalis broth culture and then an initial bacterial sample was collected from the root canal with paper cones and plated on brain–heart infusion agar. The root canals were divided into four groups as follows: Reciproc® R25 (n = 20) and Reciproc® R25 combined with Mtwo 40.04 instrument (n = 20). The negative controls consisted of five uncontaminated root canals and the positive control consisted of five contaminated roots that were not subjected to any decontamination procedure. Irrigation was performed using sodium chloride. After instrumentation, samples were collected with paper cones and the rate of bacterial reduction was calculated. Microbiological testing (colony-forming units [CFUs]) was performed to quantify the decontamination obtained by the proposed protocols. Statistical analysis was performed by paired t-test and analysis of variance test.Results:Both techniques significantly reduced the number of bacteria in the root canal (P < 0.05), with no significant difference among them (P > 0.05).Conclusion:The addition of the instrument Mtwo 40.04 after the root preparation with the instrument R25 does not improve its decontamination capability.