Sterilization in endodontic practice is a must as the major motive of an endodontist is to combat endodontic diseases by eliminating the causative microorganisms. Owing to the design of the endodontic files, it is very conducive for the microorganisms and debris to be anchored onto them. So, the aim of this study was to investigate the sterilizing efficacy of two methods of sterilization using rotary nickeltitanium files: Autoclave and chemical sterilization using two solutions (Quitanet Plus and GlutraMil) and to check whether any sterilizing technique could match the standard of autoclave and which of the two chemical sterilizing agents were more efficient. The rotary endodontic files contaminated with Bacillus stearothermophilus spore suspension, were sterilized by the two methods and their efficacy was checked by immersing the contaminated files in test tubes containing thioglycollate medium. The results of the study showed that the files sterilized by autoclave were 100% sterile and those sterilized with GlutraMil showed 75% sterility with Quitanet showing only 25% sterility. So, the study concluded that autoclave could be used as a reliable method of sterilization in clinical practice but GlutraMil and Quitanet Plus cannot be relied upon completely.