Sterilization is a process by which an article, surface or medium is freed of all microorganismsin either vegetative or spore state. Control of infection that spreads through various instruments used in endodontics is of utmost importance as a preventive measure for cross infection.The purpose of this study is to determine the effectiveness and various methods of precleaning and sterilization of endodontic files.Materials and methods: 105 used rotary ProTaper Ni-Ti files (Tulsa, DENTSPLY, U.S.A) were divided into 8 groups of 15 each and subjected to various methods of pre-cleaning and sterilization.After sterilization, the instruments were transferred into sterile test tubes containing todd-hewitt broth, incubated at 37°C for 72 hrs, and observed for bacterial growth. Results: Sterilization procedures were 100% effective for rotary files in groups B, C, D, E, and F. Rotary files used in group A,G & Control group showed evidence of bacterial growth. Conclusions: The results do not support the recommendation for the single use of endodontic files based on inability to clean files between uses. The majority of bacteria were eliminated from endodontic files after either ultrasonic cleaning or using a chemical disinfector.