Amniotic membrane is widely used in the treatment of burn wounds and ulcers of various etiology. As it comes into contact with open wounds, it needs to be perfectly sterile to avoid the transmission of any disease. Accordingly, amniotic membrane needs to bear a high sterility assurance level (SAL). Conventionally, a radiation dose of 25 kGy is the generally accepted dose for sterilization. But to keep intact the biomechanical and other properties, it is sometimes proposed to use a lower dose without compromising an SAL of 10−6. The initial microbial contamination level and the radiation resistance of the contaminants determine the dose required for sterilization. The microbial species associated with the amniotic membrane from about 70 different batches were isolated. Twenty-two representative bacterial isolates were characterized and tested for survival in an incremental series of radiation doses from 0.5 to 5.0 kGy. The radiation decimal reduction dose (D10) values for the strains were determined. Relatively higher D10 values were recorded for the gram-positive isolates. The D10 values of microbial isolates ranged from 0.16 to 1.3 kGy, and most resistant Bacillus strain had a D10 value of 2.1 kGy. The radiation dose necessary to achieve an SAL of 10−6 was calculated based on the D10values of the isolated strains. For a bioburden of 1000 Bacillus organism, the sterilization dose of 18.9 kGy is obtained. However, based on the experimental determination of D10 of the radiation-resistant reference strain Bacillus pumilus, the adequate dose for radiation sterilization is found to be 19.8 kGy if bioburden level of 1000 is granted. The results substantiate that radiation dose of 25 kGy assures sterilization of amniotic membranes with bioburden level of 1000 colony forming units.