This in vitro study examined the effect of surface defects on cutting blades on the extent of the cyclic fatigue fracture of HEROShaper Ni-Ti rotary files using fractographic analysis of the fractured surfaces. A total of 45 HEROShaper (MicroMega) Ni-Ti rotary files with a #30/.04 taper were divided into three groups of 15 each. Group 1 contained new HEROShapers without any surface defects. Group 2 contained HEROShapers with manufacturing defects such as metal rollover and machining marks. Group 3 contained HEROShapers that had been clinically used for the canal preparation of 4-6 molars. A fatigue-testing device was designed to allow cyclic tension and compressive stress on the tip of the instrument whilst maintaining similar conditions to those experienced in a clinic. The level of fatigue fracture time was measured using a computer connected the system. Statistical analysis was performed using a Tukey's test. Scanning electron microscopy (SEM) was used for fractographic analysis of the fractured surfaces. The fatigue fracture time between groups 1 and 2, and between groups 1 and 3 was significantly different (p < 0.05) but there was no significant difference between groups 2 and 3 (p > 0.05). A low magnification SEM views show brittle fracture as the main initial failure mode. At higher magnification, the brittle fracture region showed clusters of fatigue striations and a large number of secondary cracks. These fractures typically led to a central region of catastrophic ductile failure. Qualitatively, the ductile fracture region was characterized by the formation of microvoids and dimpling. The fractured surfaces of the HEROShapers in groups 2 and 3 were always associated with pre-existing surface defects. Typically, the fractured surface in the brittle fracture region showed evidence of cleavage (transgranular) facets across the grains, as well as intergranular facets along the grain boundaries. These results show that surface defects on cutting blades of Ni-Ti rotary files might be the preferred sites for the origin of fatigue fracture under experimental conditions. Furthermore, this work demonstrates the utility of fractography in evaluating the failure of Ni-Ti rotary files.