Literature has reported controversial findings on whether formalin affected bone properties, or not, especially when different preservation time durations and temperatures were involved. Hence, accurately and systematically quantifying the effect of formalin on the mechanical properties of bone using a large dataset is crucial for assessing biomechanical responses based on fixed specimens. A total of 154 longitudinal and 149 transverse cuboid-shaped (12 mm × 2 mm × 0.5 mm) specimens from the midsection of 12 bovine femora from six bovines were prepared and assigned to ten groups, including fresh-frozen, formalin-preserved at 25 °C for 4 weeks and 8 weeks, and formalin-preserved at 4 °C for 4 weeks and 8 weeks. All specimens underwent quasi-static three-point bending tests with a loading rate of 0.02 mm/s. The Young's modulus, yield stress, yield strain, tangent modulus, effective plastic strain, ultimate stress, and toughness were calculated by optimizing the material parameters to make the force-displacement curve of the finite element prediction consistent with the experimental curve, combined with specimen-specific finite element models. Preservation time and temperature both had significant effects on the Young's modulus, yield stress, effective plastic strain, yield strain and ultimate stress of cortical bone (p < 0.05). The Young's modulus, yield stress, and ultimate stress of longitudinal specimens decreased significantly with the increase of preservation time, and the yield strain increased significantly. As the preservation temperature increases, the Young's modulus of the transverse sample decreased significantly, and the yield strain increased significantly. The preservation time mainly affects the longitudinal specimens, while the preservation temperature mainly affects the transverse specimens. Formalin preservation of bovine femoral cortical bones at a lower temperature and less than 4 weeks is recommended for biomechanical testing.