Aim: To assess and evaluate the effectiveness of sterilization of endodontic files by different methods (Autoclave, Diode laser and Glutraldehyde) with and without disinfection by ultrasonics cleaner. Methodology: The study was performed on 280 K-files to test the sterilization efficacy of autoclave, glutaraldehyde and diode laser with and without ultrasonics. Standard bacterial isolates of Bacillus stearothermophilus, Enterococcus faecalis, Streptococcus mutans and fungi Candida albicans were obtained and the pre-sterilized files were contaminated. All the endodontic files randomlydivided into seven groups were subjected to sterilization by different methods of sterilization. The test tubes were kept for incubation at 55OC for 21 days and checked for growth. Presence of turbidity in a test tube indicated the presence of bacteria and that the particular file was not sterilized completely. Results: It was observed that in Group I turbidity was observed in all test tubes. No turbidity was found in any of the test tubes in Group II a and Group III a thereby indicating total sterility. The files of Group II b showed turbidity in 4 test tubes and Group III b showed turbidity in 2 test tubes (5%). Files of Group II c showed turbidity in 6 test tubes while Group III c showed turbidity in 5 test tubes. The subculture from turbid tubes of various groups were identified by various bacteriological tests. Conclusion: It can be concluded that to achieve one hundred percent of sterilization, autoclave is a suitable method for killing of all microorganisms.