The use of endodontic files multiple times can cause fatigue in them and can lead to their separation in the root canal. The purpose of this study was to achieve a reduction in cyclic fatigue stress in a newly introduced nickel-titanium (NiTi) rotary single-file system. The study aimed to determine whether cryotherapy could help reduce cyclic fatigue and stress on rotary files after multiple uses during root canal treatment. By utilizing finite element analysis (FEA), the study provided a comprehensive evaluation of how cryotherapy might enhance the performance and longevity of these instruments, ultimately benefiting patients undergoing root canal therapy. Methodology This in vitro comparative study used scanned plastic teeth with genuine root canal anatomy and FEA to investigate the mechanical response to cyclic fatigue and stress of NiTi rotary file system. The endodontic file (TruNatomy,dentsply) was created through the complex root canal geometries, for which mandibular tooth models were scanned and created by a computer software (Abaqus). The total sample size was 34, divided into two groups, with each group comprising 17 participants (n = 17). The results were analyzed by analysis of variance (ANOVA) test. Results The results revealed that the p-values were less than 0.001, indicating significant reduction in cyclic fatigue when the NiTi rotary single-file system (TruNatomy,dentsply) was treated with cryotherapy . However, stress reduction was observed in the NiTi rotary single-file system when it was treated with cryotherapy. Conclusion This in vitro comparative study concluded that cryotherapy helps to reduce the stress of NiTi rotary singlefile system. Nonetheless, more research is needed to understand the clinical significance of the findings of the current in vitro study.