Instrument separation during root canal therapy is inevitable in endodontics with several unfavorable clinical consequences. Therefore, examining the cyclic flexural fatigue resistance of commonly used rotary endodontic files is crucial. This study aimed to determine the cyclic flexural fatigue resistance of four nickel–titanium (NiTi) rotary files used as a single canal preparation technique: WaveOne, Reciproc, Protaper F2, and Unicone medium instruments. According to the manufacturer’s instructions, each file was rotated freely within a 1.3 mm deep and 1.3 mm wide V-shaped groove in a stainless-steel block with a 40° and 5 mm radius of curvature. Cyclic fatigue resistance was compared between the NiTi files by verifying the time needed to crack. The data were analyzed using one-way analysis of variance (ANOVA) followed by Scheffé post hoc with a significant level set at p < 0.05. Our results demonstrated that the WaveOne instrument had the highest cyclic flexural fatigue resistance among the tested groups (p ≤ 0.05), while Unicone had the lowest cyclic flexural fatigue resistance. This study concluded that WaveOne size 25/0.08 could illustrate a superior cyclic flexural fatigue resistance when instrumenting root canals with the lowest possibility to cause instrument separation.