Introduction: The aim of this study was to evaluate the changes in the surface profile of rotary nickel-titanium (NiTi) files designed to prepare a glide path and conventional stainless steel (SS) hand files when used in extremely narrow canals both in clinical and experimental conditions. Methods: This ex vivo/in vivo study used either SS #10 K hand files or the PathFile system (Dentsply Maillefer, Ballaigues, Switzerland) to establish a glide path in maxillary molars with narrow root canals (defined as canals that bound a #8 K-file at the working length). After treatment, all files were cleaned and scanned using a noncontact laser profilometer. Changes to the surface profile (Ra cutting edge waviness = RaCEW) and roughness (Ra cutting edge roughness = RaCER) of the cutting edges and the surface roughness of the flute area (Sa flute roughness = SaFR) were analyzed. Results: #10 K-files had significant increases in all types of measurements (RaCEW, RaCEW, and SaFR) after the preparation of second mesiobuccal canals compared with the control group (P < .05) in both studies. In the ex vivo study, the PathFile (P1) had no significant changes in the surface profile from baseline but had a significant increase in the surface roughness of the flute (SaFR) (P < .05). PathFiles that were used 3 times in vivo had a significant increase in roughness (flute and cutting edge) values across all PathFile sizes compared with control values. Conclusions: This study showed that PathFiles had significantly less surface defects compared with SS files after the preparation of narrow canals, indicating their possible benefits when establishing a glide path in extremely narrow canals.