Purpose To determine whether the bathing of an anterior cruciate ligament (ACL) autograft in vancomycin reduces the rate of infection following an ACL reconstruction. Methods Retrospective analysis of all ACL reconstructions over an 8-year period in two University Hospitals. In the initial 4-year period, all patients were operated on under classical antibiotic intravenous prophylaxis (group 1). Over the last 4-year period, this prophylaxis was supplemented with presoaking of the autograft (group 2). Presoaking was performed with sterile gauze previously saturated with a vancomycin solution (5 mg/ml). Results There were 810 and 734 patients in group 1 and 2, respectively. Fifteen cases of knee joint infections were identified in the series (0.97 %). All of these infections occurred in group 1, representing a rate of infection of 1.85 % in comparison with 0 % in group 2 ( p  < 0.001). Conclusions Autograft presoaking with vancomycin in combination with classical intravenous antibiotic prophylaxis reduced the rate of knee joint infection following an ACLR in comparison with antibiotic prophylaxis alone. This technique could be of relevance in daily clinical practice to prevent infection after ACLR. Level of evidence Case control study, retrospective comparative study, Level III.