Objective: To evaluate the bacterial extrusion during instrumentation with different nickel titanium (NiTi) engine-driven instruments. Methods: Ninety extracted single-canal human mandibular incisor teeth were inoculated with Enterococcus faecalis to obtain biofilm formation and were randomly divided to 6 groups (n=15). One group served as the control and was not instrumented; the other groups were prepared with ProTaper Gold (PTG; Dentsply Maillefer, Ballaigues, Switzerland), WaveOne Gold (WOG; Dentsply Maillefer), Twisted File Adaptive (TFA; SybronEndo, Orange, CA, USA), One Shape New Generation (OSNG; MicroMega, Besancon, France), and K3XF (SybronEndo) instruments. Bacteria extruded beyond the apical foramen were quantified in colony-forming units per milliliter. The number of colony-forming units in the remaining biofilm was determined for each sample. Data were analyzed using the one-way analysis of variance (ANOVA) and Tukey post-hoc tests.