Objective: To compare the efficacy of manual and mechanical instrumentation techniques, including ProTaper Universal retreatment system, Mtwo retreatment system, Reciproc system, and Hedström files, regarding removal of overextended root canal filling material. Materials and Methods: Eighty extracted human mandibular premolar teeth were prepared at the apical foramen level using Revo-S rotary files and subsequently obturated. The root canal filling material was deliberately extruded from the apex. Samples were transferred to glass vials that simulated the periapical area. Eighty samples of overfilled teeth were randomly assigned to four equal groups (n = 20) for removal of the root filling material with ProTaper Universal retreatment files (Group 1), Mtwo retreatment files (Group 2), Reciproc system (Group 3), and hand files (Group 4). Removal of the root canal filling material and additional preparation were performed by individual instruments from each different system up to a #40 size. The external apical surface of the teeth and the surrounding glass vials were checked using a dental operation microscope with ×12.5 magnification. Samples were divided into two groups based on whether removal of the overextended root canal filling material was successful or not. The Fisher's exact test was used to detect any significant difference between the groups (α = 0.05). Results: The success rate for removal of overextended gutta-percha was greater for the Mtwo (30%) and hand files (30%) compared with the ProTaper (20%) and Reciproc (10%). However, no significant statistical differences existed among the experimental groups (P > 0.05). Conclusions: This study demonstrated that all tested systems had similar efficacy in removing overextended root canal filling material.