Purpose: To present the clinical and imaging results of a series of patients undergoing anterior cruciate ligament reconstruction with vancomycin presoaking of the hamstring autograft compared with patients in the immediate period prior, when no vancomycin was used. Methods: This was a retrospective sequential series of patients with anterior cruciate ligament reconstruction using either a graft protocol with no vancomycin presoaking (group 1, January 2013 to October 2015) or a graft protocol with vancomycin presoaking (group 2, November 2015 to December 2018). Lysholm and International Knee Documentation Committee scores were obtained at a minimum 24-month follow-up. Graft ruptures were recorded. Between 6 and 12 months’ follow-up, magnetic resonance imaging (MRI) was obtained to evaluate graft healing and integration. Results: There were 102 patients (72% male patients), with 40 in group 1 (mean age, 32.2 years) and 62 in group 2 (mean age, 32.3 years). A graft rupture occurred in 5 patients (13%) in group 1 and 6 patients (10%) in group 2 (P = .65). The median Lysholm score was 95 points (interquartile range [IQR], 86-100 points) in group 1 and 95 points (IQR, 90-100 points) in group 2 (P = .37). The median International Knee Documentation Committee score was 93 points (IQR, 82-99 points) in group 1 and 94 points (IQR, 86-99 points) in group 2 (P = .22). MRI evaluation of integration showed that 87 patients (90%) had no synovial fluid at the tunnel-graft interface, without a difference between groups (P = .24). On the basis of graft signal appearance, hyperintense grafts were found in 45 patients (46%); isointense, 45 (46%); and hypointense, 7 (7%). Group 1 had a higher prevalence of hypointense grafts, whereas group 2 had a higher prevalence of hyperintense and isointense grafts (P = .003). Conclusions: Vancomycin presoaking of hamstring grafts increased the number of hyperintense and isointense grafts on MRI. Additionally, more hypointense grafts were noted when vancomycin was not used, suggesting the presence of more mature grafts in the non-vancomycin group. Level of Evidence: Level III, retrospective comparative study.