The main aim of this study is the estimation of microbial load on tracheostomy tube and subsequent optimization of gamma radiation dose for efficient decontamination of the tube. The microflora of previously EtO sterilized tracheostomy tubes of three different companies was ascertained. The microflora was then identified by morphological analysis of the bacterial colonies followed by Gram staining and biochemical characterization tests. These tests revealed the presence of two different kinds of gram positive bacteria: Bacilus subtilis and Staphylococcus epidermidis. Three samples of the company having maximum microbial load were then selected for dose optimizaion of gamma radiation. The samples were sent to Pakistan Radiation Services (PARAS) for gamma radaition treatment at selected doses of 20 kGy, 25 kGy and 30 kGy. In this study effective elimination of bioburden was achieved at 30 kGy, although 25 kGy is recommended as the standard dose for sterilization of medical devices. This study reveals the importance of proper sterilization of tracheostomy tube to prevent patients from secondary infections thereby improving health standards.