In this study, poly(methacrylic acid-glycine)-grafted agar-based hydrogels with optimized process parameters were synthesized via a two-step green-radiation induced grafting process using microwave heating. Poly(methacrylic acid) chains were graft copolymerized onto an agar backbone using ammonium persulfate as a free radical initiator and N,N'-methylene-bis-acrylamide as a cross-linking means using microwave heating. The influence of different reaction parameters was investigated on the percentage swelling behavior of the cross-linked hydrogel networks. The prepared hydrogel networks with optimum percentage swelling were characterized by Fourier transform infrared spectroscopy, time-of-flight secondary ion mass spectrometry, scanning electron microscopy, X-ray diffraction, and thermogravimetric analysis, using agar as a reference. The anti-bacterial activities of the prepared hydrogels against Gram-positive Staphylococcus aureus bacteria and Gram-negative Escherichia coli bacteria were investigated. Staphylococcus aureus was found to be more susceptible to the compounds compared to Escherichia coli. These results indicate that the prepared hydrogels have the potential to be applied as anti-bacterial agents.