Anterior cruciate ligament ACL reconstruction using the double-bundle (DB) technique is gaining popularity. A possible weak link in the DB technique could be that two tendon grafts of smaller diameters are used. The purpose of this study was to test different femoral fixation methods and graft diameters representing single-bundle (SB) and DB ACL reconstructions and compare their biomechanical properties. We hypothesized that SB 6-mm graft constructs had inferior biomechanical properties than SB 9-mm grafts or DB 2 × 6-mm grafts. Furthermore, we hypothesized that interference (IF) screw fixation would demonstrate less elongation and a higher stiffness than Endobutton (Smith & Nephew®, Inc., Andover, Massachusetts, USA) fixation (EBF). We performed an in vitro study using porcine knees and extensor tendons. The mechanical test consisted of a cyclic test followed by a load-to-failure test. We found that 6-mm graft constructs had an ultimate failure load that was up to 40% less than both the 9-mm and 2 × 6-mm graft constructs, despite the fixation method (P-values ≥ 0.004). Comparing fixation methods, EBF was superior to IF concerning maximum load to failure (P < 0.001); IF resulted in a higher stiffness of the femur/graft complex than the EBF (P < 0.001) but no significant difference in elongation between fixation methods. Since the two graft strands are subjected to different loads in different knee flexion angles, the reduced strength of the individual graft strands in DB ACL reconstruction could be a concern.