Anterior cruciate ligament (ACL) injuries are one of the most common acute sports injuries in male football. Recently, the role of muscle fiber type (MFT) distribution in both athletic performance and hamstring muscle injury risk has gained popularity in sports medicine. As the hamstring muscle unit is a biomechanical synergist of the ACL, this study intended to verify to what extent hamstring MFT distribution is related to ACL injury in football players, using proton magnetic resonance spectroscopy (1H-MRS). Thirty-seven male football players with a recent history of ACL injury and 49 matched controls were submitted to 1H-MRS evaluation of the hamstrings. MFT distribution was estimated based on the relative carnosine content. Results demonstrated that carnosine contents differed significantly based on ACL history presence, with higher carnosine content in the injury group. Average carnosine contents of 0.204 ± 0.011 and 0.169 ± 0.008 AU were seen in the ACL injury and control groups, respectively (p = 0.011). FT, IT, and ST fiber dominances were estimated to be 27%, 46%, and 27% in the ACL group and 18%, 43%, and 39% in the control group. ACL injury history appears to present a significant association with hamstring muscle carnosine content, suggesting higher FT fiber dominance might be a consequence or a cause. Future prospective research is needed to verify to what extent intramuscular carnosine content is causally related to (non-contact) ACL injury risk. In case of prospective confirmation, MFT determination might be valuable in injury prevention and load management strategy development.