PURPOSE: Inadvertent contamination of the hamstring autograft during ACL reconstruction is infrequent, but can result in significant complications. The purpose of this study is to evaluate bacterial contamination of hamstring autografts dropped onto the operating room floor and methods of graft decontamination. METHODS: Hamstring tendons were harvested from patients. Excess tendon not used in the ACL procedure was divided into 6 segments. Segments were assigned to 6 groups (A through F, N = 30 in each group): group A: uncontaminated graft immediately postharvest (control), group B: graft dropped onto the floor (5 s), group C: graft dropped onto the floor (15 s). grafts in groups D to F were dropped onto floor for 15 s then rinsed with saline (group D), bacitracin solution (group E) or chlorhexidine 4 % solution (group F) for 3 min. All grafts were sent to the microbiology laboratory for anaerobic and aerobic cultures. RESULTS: Cultures were positive in 23 % of graft segments from group A (7/30), 33 % of grafts from group B (10/30), 23 % from group C (7/30), 30 % from group D (9/30) and 3 % from both group E (1/30) and group F (1/30). Sixteen unique organisms were identified, with Staphylococcus aureus as the most common isolate. Grafts rinsed in either bacitracin solution or 4 % chlorhexidine solutions were significantly less likely to be culture positive when compared to control graft segments (p < 0.05). However, there was no significant difference between uncontaminated grafts retrieved in <5 versus 15 s from the floor. CONCLUSION: This study supports the practice of decontaminating a dropped ACL hamstring autograft using either 4 % chlorhexidine or bacitracin solution. Specimens should be retrieved sterilely and washed for at least 3 min. This study also demonstrates no advantage in retrieval time of less than 5 s as compared to 15 s for uncontaminated graft. Hamstring harvest in ACL reconstruction may result in positive cultures, thus routine soaking of the hamstring autograft in either bacitracin or 4 % chlorhexidine solution is recommended. In addition, dropped hamstring autograft can be effectively sterilized with bacitracin or 4 % chlorhexidine solution. LEVEL OF EVIDENCE: II.