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. Results: All NiTi instruments resulted in different quantities of bacterial extrusion. The TFA group caused most bacterial extrusion (p<0.05). The PTG and WOG groups caused less bacterial extrusion than the OSNG and K3XF groups (p<0.05), but there was no statistically significant difference between the PTG and WOG groups (p>0.05). Conclusion: PTG and WOG are preferable system in terms of successful endodontic treatments. The amount of bacterial extrusion is associated with the metallurgy and design of the instrument used.