The purpose of this in vitro study is to examine the processing of the surfaces of the files after removing the gutta percha in the root canals with different file systems. Eighteen mandibular molar teeth were used. The access cavities were prepared, the mesiobuccal canals were shaped and filled with the lateral condensation. During filling, AH Plus and Bioceramic sealers were used. The root canal fillings were removed using ProTaper Universal Retreatment (D1, D2, D3), Reciproc Blue file (R40), and TF Adaptive rotary file (SM2). Surface changes of the files after the removal of the root canal filling material in the canals were examined under Atomic Force Microscope (AFM) and Scanning Electron Microscope (SEM). The analysis revealed that the type of sealer used, whether resin or bioceramic, did not have a significant impact on the surface of the files (p > 0.05). ProTaper Universal Retreatment (PTUR) instruments showed significantly less Ra and RMS values compared to the other files. Additionally, a significant difference was found between RCP Blue and TFA files in Ra and RMS values (p < 0.05). PTUR instruments demonstrated better surface values regardless of the root canal sealer.