Reported infection rates following anterior cruciate ligament (ACL) reconstruction are low, but infections are associated with high morbidity including reoperations and inferior clinical outcomes. The purpose of the current study was to investigate the rate of infection after ACL reconstruction with and without graft preparation with a vancomycin irrigant. All ACL reconstructions performed between May 2009 and August 2018 at a single academic institution were reviewed and categorized based on vancomycin use. Patients with <90-day follow-up, intraoperative graft preparation with an antibiotic other than vancomycin, or previous ipsilateral knee infection were excluded. Infection was defined as a return to the operating room for irrigation and debridement within 90 days after ACL reconstruction. Descriptive and inferential statistical analysis using t tests and Poisson regression were performed, with significance defined as p < 0.05. In total, 1,640 patients (952 males; 58.0%) with a mean age (and standard deviation) of 27.7 ± 11.4 years underwent ACL reconstruction (1,379 primary procedures; 84.1%) and were included for analysis. Intraoperative vancomycin was used in 798 cases (48.7%), whereas 842 ACL reconstructions (51.3%) were performed without intraoperative vancomycin. In total, 11 reconstructions (0.7%) were followed by infection, which occurred in 10 (1.2%) of the patients in whom the graft was not soaked in vancomycin and in 1 (0.1%) of the patients in whom the graft was soaked in vancomycin (p = 0.032). Age (p = 0.571), sex (p = 0.707), smoking (p = 0.407), surgeon (p = 0.124), and insurance type (p = 0.616) were not associated with postoperative infection risk. Autograft use was associated with decreased infections (p = 0.045). There was an 89.4% relative risk reduction with the use of intraoperative vancomycin. An increased body mass index (BMI) (p = 0.029), increased operative time (p = 0.001), and the absence of ACL graft preparation with vancomycin (p = 0.032) independently predicted postoperative infection. The use of vancomycin-soaked grafts was associated with a 10-fold reduction in infection after ACL reconstruction (0.1% versus 1.2%; p = 0.032). Other risk factors for infection after ACL reconstruction included increased BMI and increased operative time. Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.