Present studies were conducted to estimate the bioburden level of human amniotic membranes; and to detect inhibitory effects of antibiotic and radiation on the bacterial flora associated with amniotic membrane. Bacterial counts per gram of the samples (25) were found 5.42×102 to 8.87×106 CFU/gm. Fifteen different colonies were isolated from the samples and identified up to the genus level based on several morphological, physiological and biochemical characteristics where the isolates were identified as Bacillus sp. (5), Staphylococcus sp. (3), Micrococcussp. (2), Pseudomonas sp. (2), Achromobacter sp. (1), Alcaligenes sp. (1) and Citrobacter sp. (1). In vitro Inhibitory activities of three different antibiotics were screened against the isolates where all isolates were found sensitive to Gentamicin. ExceptAlcaligenes sp. and Achromobacter sp., all isolates were found resistant to Ampicillin. Similarly all isolates were also found resistant to Cloxacillin except Micrococcus sp. and Pseudomonas sp. Isolates were treated in an incremental series of γ-radiation doses from 2.5-25 kGy where all Gram positive bacteria were killed at 15 kGy. Gram negative bacteria showed more sensitivity than the positives and were killed at 5 kGy. Based on these studies it could be concluded that antibiotic decontamination should not be regarded as the way of obtaining sterility due to resistance of bacteria against antibiotics. Gamma-radiation assures complete sterility.