Aim: The study aims to compare and evaluate the fracture resistance of endodontically treated bicuspids instrumented with Hand Files, TruNatomy (TRN), ProTaper Next (PTN), ProTaper Gold (PTG), and WaveOne (WO). Materials and Methods: In total, 45 extracted single-rooted human premolar teeth were selected and sectioned at or below the cementoenamel junction to obtain roots 15 mm long. The samples were divided into four experimental groups and one control group ( n = 9): instrumentation with nickel–titanium (NiTi) Hand Files (control group), instrumentation with TRN files, instrumentation with PTN files, instrumentation with PTG files, and instrumentation with WO files. The samples were obturated by lateral compaction technique using gutta-percha points and AH Plus sealer. A vertical load was applied to the tooth using the universal testing machine (Tecsol-TSI-BDS-2Kn-Sr No. 170710) for the fracture resistance test. Statistical analysis was performed. Results: The study reported that teeth instrumented with NiTi hand files exhibited the highest fracture resistance when compared to all the rotary and reciprocating file systems. Among rotary and reciprocating instruments, root prepared with TRN files showed the most significant resistance to fracture compared with PTN, PTG, and WO files. Conclusion: Within the limitations of this study, it can be concluded that minimally invasive instrumentation of the system preserves more pericervical dentin, which may increase the resistance to fracture. Results of this study and other studies support the use of minimally tapered instruments to improve the fracture resistance of endodontically treated teeth.