Aims. Graft infection following anterior cruciate ligament reconstruction (ACLR) may lead to septic arthritis requiring multiple irrigation and debridement procedures, staged revision operations, and prolonged courses of antibiotics. To our knowledge, there are no previous studies reporting on how gentamicin pre-soaking of hamstring grafts influences infection rates following ACLR. We set out to examine this in our study accordingly. Methods. This retrospective study included 2,000 patients (1,156 males and 844 females) who underwent primary ACLR with hamstring autografts between 2007 to 2017. This included 1,063 patients who received pre-soaked saline hamstring grafts for ACLR followed by 937 patients who received pre-soaked gentamicin hamstring grafts for ACLR. All operative procedures were completed by a single surgeon using a standardized surgical technique. Medical notes were reviewed and data relating to the following outcomes recorded: postoperative infection, clinical progress, causative organisms, management received, and outcomes. Results. Superficial wound infection developed in 14 patients (1.31 %) receiving pre-saline soaked hamstring grafts compared to 13 patients (1.38 %) receiving pre-gentamicin soaked hamstring grafts, and this finding was not statistically significant (p = 0.692). All superficial wound infections were treated with oral antibiotics with no further complications. There were no recorded cases of septic arthritis in patients receiving pre-gentamicin soaked grafts compared to nine patients (0.85%) receiving pre-saline soaked grafts, which was statistically significant (p = 0.004). Conclusion. Pre-soaking hamstring autographs in gentamicin does not affect superficial infection rates but does reduce deep intra-articular infection rates compared to pre-soaking hamstring grafts in saline alone. These findings suggest that pre-soaking hamstring autografts in gentamicin provides an effective surgical technique for reducing intra-articular infection rates following ACLR. Cite this article: Bone Jt Open 2021;2(1):66–71