Abstract Background Failures to follow recommendations for reprocessing of surgical instruments may place patients at risk for exposure to pathogenic microorganisms. When such failures occur, medical facilities often face considerable uncertainty and challenges in assessing the actual risks of disease transmission. Methods In 2011, staff at an Ohio hospital determined that surgical instruments inside a Steriset Container had inadvertently been autoclaved on a gravity cycle rather than on the recommended pre-vacuum cycle, potentially exposing 72 patients who underwent surgery with the instruments to risk of infection. To provide an assessment of the level of risk, we tested the effectiveness of the machine washer/disinfector step and of the sterilization process inside the Steriset Container on the gravity cycle for killing of Geobacillus stearothermophilus spores, Clostridium difficile spores, and methicillin-resistant Staphylococcus aureus (MRSA). Based on the test results, the risk of transmission of MRSA by the instruments was calculated and the risk of transmission of hepatitis B virus was estimated. Results The machine washer/disinfector consistently reduced MRSA recovery by a factor of 1:100,000. The sterilization process inside the Steriset Container consistently reduced MRSA concentrations by a factor of >1:10,000,000 and killed 105 C. difficile spores and 105 G. stearothermophilus spores. The risk of MRSA transmission due to the incident was calculated to be 1 in 100 trillion. Conclusions The risk for transmission of infection due to the failure to follow recommended sterilization processes was negligible based upon complete killing of G. stearothermophilus biological indicator spores, C. difficile spores, and MRSA under conditions that replicated the incident where proper procedures were not followed. Such real-time assessments of the risks associated with specific incidents may provide evidence-based information that can be used to inform decisions regarding disclosure of the incident to patients.