One of the primary goals in endodontics is efficient cleaning and shaping of the root canal while maintaining its original configuration. Numerous studies highlight the advantage of using nickel-titanium instruments and the most important is their high flexibility and the ability to penetrate the curved canals. The aim of this study was to analyze the ultrastructure of the surface of nickel-titanium endodontic files before and after use. Eighty-four instruments were included in this study. The instruments were used one, three and six times for root canal preparation on extracted upper premolars. The analysis was performed based on the number of uses and the size of the instruments. Changes on flat surfaces in the grooves of the instruments, defects of the cutting edges and fractures were evaluated. The surface ultrastructure was analyzed on a scanning electron microscope (SEM). SEM analysis showed irregularities on the surfaces of the new instruments. Surface defects and damage of the cutting edges were present after the first use. After three uses, the damage was more evident in instruments of all sizes. After six uses, all instruments showed damage of the cutting edges, and a significant number of fractures were observed. Irregularities on the working parts are already noticed on the new endodontic instruments and originate from the production process. The first signs of damage appear after the first use. In order to prevent errors in clinical work during root canal preparation, it is recommended that the number of instruments uses should be limited.