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Atomic force microscopy (AFM) is a well established and documented tool for materials investigation. The aim of this study was to evaluate the topography of conventional stainless-steel files and both hand and rotary nickel-titanium (NiTi) files by using AFM. One endodontic file of each of the following was selected: stainless-steel K-file Dentsply, stainless-steel K-file Moyco, hand NiTi K-file Nitiflex, hand NiTi Greater Taper, rotary NiTi Greater Taper, and rotary NiTi Quantec. The analyses were performed on twenty different points located along a 3-mm section starting at the tip of each file. Root mean square (RMS) parameters for contact mode imaging microscopy variations were measured. The differences between RMS values were tested by ANOVA with Fisher's protected LSD test for multiple comparisons (p < 0.05). RMS of depth profile data was used to determine any statistically significant difference in vertical amplitude. According to results, all instruments showed topographic irregularities distributed on surface. Endodontic files manufactured by the same method and alloy demonstrated significant differences, whereas no significant differences were found for instruments produced by different alloys and methods (p < 0.05). The hand NiTi Greater Taper, rotary NiTi Greater Taper, and rotary NiTi Quantec showed greater values of vertical amplitude topography compared to K-Dentsply and Nitiflex files (p < 0.05). The AFM technique proved to be a valuable research tool in the investigation of endodontic files topography.