Introduction Enterococcus faecalis (E. faecalis) is the most commonly isolated bacterium from infected root canals. It is found in the form of a biofilm, which makes it more resistant to antimicrobials, and requires optimal chemomechanical strategies to maximize root canal disinfection. Aim To evaluate the efficacy of 4 different endodontic file systems against E. faecalis biofilm growth in root canals using colony-forming units per milliliter (CFU/mL) and scanning electron microscope (SEM). Methods Eighty-five extracted human mandibular premolars with straight root canals and apical diameters not larger than the #15 K-file were randomly selected. After performing a pilot study (n = 15) to determine the ideal incubation period for E. faecalis biofilm development, sixty-five root canals were infected with E. faecalis, incubated for 3 weeks, and then mechanically prepared using one of four single files (XP-endo Shaper, Hyflex EDM, One Curve, and Fanta. AFTM F One) (n = 15). Five infected root canals were excluded for the positive control. Five non-contaminated root canals were included for the negative control. Samples were collected using sterile paper points pre- and post-instrumentation to determine the bacterial load (CFU/mL). Root canals from each group were topographically evaluated at the coronal, middle, and apical segments using scanning electron microscope (SEM). Bacterial reduction data were estimated and statistically analyzed by Kruskal–Wallis and Mann–Whitney U tests (post hoc test) (P ≤ .05). Results XP-endo Shaper, Hyflex DEM, and One Curve significantly could eradicate E. faecalis biofilms in infected root canals with no significant difference among them compared to Fanta. AF™ F One. Conclusion None of the systems were capable of completely eliminating biofilms. XP-endo Shaper, Hyflex EDM, and One Curve mechanically eliminated E. faecalis biofilms compared to Fanta. AF™ F One from infected root canals.