Abstract The aim of this study was to undertake a qualitative and quantitative assessment of nanoscale alterations and wear on the surfaces of nickel-titanium (NiTi) endodontic instruments, before and after use, through a high-resolution, noncontact, three-dimensional optical profiler, and to verify the accuracy of the evaluation method. Cutting blade surfaces of two different brands of NiTi endodontic instruments, Reciproc R25 (n = 5) and WaveOne Primary (n = 5), were examined and compared before and after two uses in simulated root canals made in clear resin blocks. The analyses were performed on three-dimensional images which were obtained from surface areas measuring 211 × 211 µm, located 3 mm from their tips. The quantitative evaluation of the samples was conducted before and after the first and second usage, by the recordings of three amplitude parameters. The data were subjected to statistical analysis at a 5% level of significance. The results revealed statistically significant increases in the surface wear of both instruments groups after the second use. The presence of irregularities was found on the surface topography of all the instruments, before and after use. Regardless of the evaluation stage, most of the defects were observed in the WaveOne instruments. The three-dimensional technique was suitable and effective for the accurate investigation of the same surfaces of the instruments in different periods of time.