Chitosan holds net ionic positive charges, which contribute its ability to chemically bind with negatively charged fats, lipids, metal ions, proteins, and microorganisms. Magnesium oxide (MgO) nanoparticles are important inorganic materials with a wide band-gap used in many applications such as catalysts, antibacterial and medical products. The aim of this study was to investigate the effect of Chitosan (CHT) hydrogel loaded MgO nanoparticles on the bacterial growth. CHT/ poly vinyl alcohol (PVA)/ poly ethylene glycol (PEG) hydrogel was blended with various amounts of MgO nanoparticles. The surface morphology of the obtained blends was investigated by Field Emission Scanning Electron Microscope (FE-SEM). Evidently, surfaces with appropriate roughness were obtained for most of the prepared hydrogels. Fourier Transform Infrared Spectroscopy (FT-IR) and Energy Dispersive X-Ray analysis (EDX) were also included in this paper. Thermal properties of all samples was studied by DSC-TGA curves. The antibacterial activity of the prepared hybrids CHT/PVA/PEG/MgO nanoparticles have performed against gram positive bacteria Staphylococcus aureus (S.aureus) and Streptococcus, as well as gram negative bacteria Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E.coli). In this study, MgO nanoparticles various proportions presented high efficiency towards gram positive microorganisms. High resistance of gram negative bacteria against the final products was extremely detected according to measured inhibition zones which were between (0-9) mm.