ObjectivesThe impact of conservative instrumentation on the disinfection of root canals with different curvatures has not yet been determined. This ex vivo study aimed to evaluate and compare the effect of conservative instrumentation with TruNatomy (TN) and Rotate and a conventional rotary system, ProTaper Gold (PTG), on root canal disinfection during chemomechanical preparation of straight and curved canals.Materials and methodsNinety mandibular molars with straight (n= 45) and curved (n= 45) mesiobuccal root canals were contaminated with polymicrobial clinical samples. Teeth were divided into three subgroups (n= 14) according to the file systems and the curvature. Canals were instrumented with TN, Rotate, and PTG, respectively. Sodium hypochlorite and EDTA were used as irrigants. Intracanal samples were taken before (S1) and after (S2) instrumentation. Six uninfected teeth were used as negative controls. The bacterial reduction between S1 and S2 was measured by ATP assay, flow cytometry, and culture methods. Kruskal–Wallis and ANOVA tests were followed by the Duncan post hoc test (p< 0.05).ResultsBacterial reduction percentages were similar for the three file systems in straight canals (p> 0.05). However, PTG showed a lower reduction percentage of intact membrane cells in flow cytometry than TN and Rotate (p= 0.036). For the curved canals, no significant differences were obtained (p> 0.05).ConclusionConservative instrumentation of straight and curved canals using TN and Rotate files resulted in similar bacterial reduction compared to PTG.Clinical relevanceThe disinfection efficacy of conservative instrumentation is similar to conventional instrumentation in straight and curved root canals.