BACKGROUND: Accidental contamination of anterior cruciate ligament (ACL) grafts during surgery is a relatively unexplored issue. This study aims to evaluate bacterial contamination in ACL grafts and the effectiveness of Microdacyn® in reducing contamination. The species of bacteria present on contaminated grafts and their antibiotic susceptibility were also analyzed. METHODS: A total of 70 patients undergoing ACL reconstruction between 2019 and 2022 were included. Patients with prior knee surgery or immunodeficiency were excluded. Grafts were divided into three groups: (1) control (kept sterile), (2) contaminated (dropped on the operating room floor), and (3) rinsed (dropped and then decontaminated in Microdacyn®). Bacteriological examination and drug sensitivity tests were performed on all grafts. Statistical analysis was conducted using chi-square tests. RESULTS: Grafts dropped on the floor showed a 74.3% (52 out of 70) contamination rate, with Staphylococcus epidermidis being the most commonly observed bacteria. After decontamination with Microdacyn®, 65.7% (46 out of 70) of the grafts were found sterile. The most sensitive antibiotics were trimethoprim + sulfamethoxazole, aztreonam and ceftazidime (97% effectiveness), while vancomycin, gentamicin, and meropenem were 95-96% effective. CONCLUSIONS: While Microdacyn® reduced bacterial contamination, a significant portion of grafts remained contaminated, indicating that it is not fully effective. However, the bacteria found on the grafts showed high susceptibility to simple and inexpensive antibiotics, which did not contribute to increased resistance. Further studies exploring alternative decontamination methods are recommended to reduce the risk of complications from graft contamination during ACL reconstruction. LEVEL OF EVIDENCE: II.