Background: Not only favorable disinfection and sterilization are inevitable in terms of medicine, but are crucial in controlling costs and reducing mortality.Objective: The aim of this study was to compare face-to-face and distance learning methods on knowledge, attitude and practice of washing and disinfection of surgical instruments among operating room staff.Methods: This interventional study was conducted on 60 operating room staff that were allocated to three groups (control, face-to-face learning and distance learning) in 2012. All 3 groups completed a self-developed questionnaire to assess knowledge, attitude and practice of washing and disinfection of surgical instruments before, one month and two months after intervention. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests.Findings: One month after intervention, the differences between three groups were statistically significant in terms of knowledge, attitude. Two months after intervention, the differences between three groups were statistically significant in terms of knowledge, attitude and practice. Two months after intervention, mean scores of knowledge, attitude and practice was increased significantly in face-to-face learning group compared to other groups.Conclusion: Face-to-face learning had a great influence on knowledge, attitude and practice of washing and disinfection of surgical instruments in operating room staff.