Background: There are limited data detailing the appropriate management of nondisposable autologous osteoarticular fragments that have been contaminated by the operating room floor. The goal of the present study was to perform a comprehensive, three-phase investigation to establish an appropriate intraoperative algorithm for the management of the acutely contaminated, but nondisposable, autologous osteoarticular bone fragment. Methods: Phase I of the study was performed to quantify the rate of contamination and microbial profile of human osteoarticular fragments that were dropped onto the operating room floor (n = 162). Phase II was performed to assess the feasibility and optimal means of decontaminating 340 similar fragments that underwent controlled contamination with bacteria that were identified in Phase I; decontamination was performed with use of cleansing agents that are routinely available in an operating room. Phase III was performed to assess the effect of each decontamination process on fragment chondrocyte viability through histologic evaluation. Results: The contamination rate in Phase I was 70%. Coagulase-negative Staphylococcus was the most commonly cultured organism. In Phase II, varying exposure time to the chemical agents did not make a significant difference in decontamination rates. Mechanical scrubbing was superior to mechanical saline solution lavage (zero of fifty-six cultures compared with twenty of fifty-six cultures were positive for coagulase-negative Staphylococcus; p < 0.001). As a whole, bactericidal agents were found to be more effective decontaminating agents than normal saline solution. Povidone-iodine and 4% chlorhexidine gluconate were the most effective decontaminating agents, with none of the twenty-eight specimens that were decontaminated with each agent demonstrating positive growth on culture. Phase III demonstrated that the groups that were treated with normal saline solution and povidone-iodine retained the greatest number of live cells and the least number of dead cells. Mechanical scrubbing significantly decreased chondrocyte viability as compared with a normal saline solution wash (p < 0.05). Conclusions: The majority of osteochondral fragments that contact the operating room floor produce positive bacterial cultures. Five minutes of cleansing with a 10% povidone-iodine solution followed by a normal saline solution rinse appears to provide the optimal balance between effective decontamination and cellular toxicity for dropped autologous bone in the operative setting. Clinical Relevance: This study provides guidance on treatment of dropped osteoarticular fragments during surgery in order to decrease the rate of contamination and infection.