Summary: A series of poly vinyl alcohol (PVA)/starch hydrogel membranes (0gm, 0.1gm, 0.3gm, 0.5gm, 0.7gm, 1gm, 1.5gm) were prepared with varying contents of piper nigrum and cinnamomum verum by solution casting method. The comparison was made between the pure PVA/starch hydrogel and natural antimicrobial agents assembled hydrogel matrix membrane. Glutaraldehyde was added as a crosslinker. Whereas, Glycerin was incorporated as a plasticizer to increases the ductility of hydrogels membranes. The concentration of loaded natural agents affects the physical properties of the hydrogel determined by swelling behavior, gel fraction, moisture retention and water vapor transmission rate. The prepared membranes were characterized by scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR). SEM shows saturated and matrix structure of membrane which makes the tensile strength of fabricated membranes as high as 9.8 MPA, which suitable to be used in biomedical applications. Antimicrobial activity was investigated against Escherichia coli and methicillin-resistant Staphylococcus aureus bacterial strains. The anti-fungal activity of these membranes was studied for Aspergillus Flavus, Aspergillus Oryzae and Aspergillus Subolivaeus fungus strains. Hydrogel membrane with 1.5g of natural antimicrobial agents has shown greater inhibitory activity. The results predict that; the membrane can be used as a wound dressing to provide a cushioning effect on the skin. © 2019 Chemical Society of Pakistan. All rights reserved.