Background and Objective: Human amniotic membrane, which is the innermost layer of placenta, contains beta defensins and elafin, which both have antibacterial properties. These antibacterial activities depend on the conditions and environmental factors. The purpose of this study was to evaluate the effect of environmental factors on human amniotic membrane antibacterial properties against Klebsiella pneumonia (ATCC7881), Entrococcus faecalis (ATCC29212), Pseudomonas aeruginosa (ATCC27853), Salmonella enteric (BAA-708), and E. coli (ATCC25922) strains in vitro. Materials and Methods: The amniotic membrane samples were obtained from caesarean women in Imam Khomeini hospital. Participating women were all seronegative for HIV, HBV, HCV, and syphilis. The samples were cut into 1.5×1.5 cm pieces. The 0.5 McFarland bacterial suspensions were prepared and spread on Muller-Hinton agar medium, and a piece of membrane was placed in the centre of each plate. Samples were examined at different time intervals (24, 48, and 72 hr), temperatures (25, 33, 37°C) and pH (6.5, 7, and 7.5) as variables. Results: The results show that time and pH as variable parameters did not affect the antibacterial properties of the amniotic membrane. However, the change in temperature (25 and 33°C vs. 37°C) had a significant impact on P. aeruginosa. Conclusion: Antibacterial properties of the amniotic membrane seem resistant against environmental factors, except for especial cases, and this sustainability could expand its usage in clinical procedures and different conditions.