This study aimed to test and compare the biomechanical properties of three tibial fixation methods of anterior cruciate ligament (ACL) tendon grafts under cyclic load and load-to-failure testing in the bovine proximal tibiae, comprising (1) staple fixation alone, (2) interference screw fixation alone, and (3) interference screw fixation with a supplementary staple. Twenty-four bovine tibiae used in the study were divided into three groups (eight proximal tibiae in each group) based on tibial fixation methods of ACL tendon grafts: group A (a spiked ligament staple alone), group B (a cannulated interference screw alone), and group C (a cannulated interference screw with a supplementary staple). Each graft fixation was exposed to cyclic loading conditions. Significant differences were determined in failure load among the three groups (p = 0.008). The mean failure load was significantly higher in group B (717.04 ± 218.51 N) than in group A (308.03 ± 17.22 N) (p = 0.006). No significant differences were observed among the groups regarding axial stiffness (p = 0.442). Cyclic displacement differed significantly among the three groups (p = 0.005). In pairwise comparisons, the mean cyclic displacement was significantly higher in group A (8.22 ± 3.24 mm) compared with group C (1.49 ± 0.41 mm) (p = 0.005). Failure displacement varied considerably among the groups (p = 0.037). Although group B (15.53 ± 6.43 mm) exhibited a greater mean failure displacement than both group A (4.9 ± 0.75 mm) and group C (8.84 ± 4.65 mm), these differences did not reach statistical significance (p = 0.602 and p = 0.329, respectively). Interference screw fixation alone and supplementary staple fixation have biomechanically similar characteristics in terms of initial strength and stiffness of tibial ACL soft tissue graft fixation. Regardless of staple use, an interference screw with the same diameter as the tibial tunnel can ensure sufficient tensile strength in tibial ACL graft fixation.