The antibacterial activity of Whey Milk and its fractions were studied against one hundred bacteria strains isolated and screened from various patients at the University Hospital of Zagazig, Sharkia Province, Egypt. F2 fraction of Whey Milk has shown the highest degree of antibacterial activity against the pathogenic bacteria A63 isolated from the wound. Buffalo whey (BW) was hydrolyzed with papain followed by fractionation using size exclusion chromatography (SEC). The fractionation profile of BWH showed three major peaks in Size exclusion chromatography, SECF1, SECF2, and SECF3 after 3h. F2 is the most active fraction and has faster migration than HP, F1, and F3 and may have higher positive charges on Urea-PAGE towards the cathode direction. FTIR spectrum analysis of the F2 fraction of whey protein demonstrates the occurrence of distinctive functional groups, such as Alkanes, alcohols, phenols, flavonoids, alkenes, anhydrides, and halo compounds. The biochemical characters and the 1.5 Kb 16S RNA gene’s nucleotide sequence of the most potent stain showed 99% similarity with Staphylococcus pasteuri. Our results showed that the ability of nanoparticles of whey to inhibit pathogenic bacteria decreases compared to raw whey and its fractions. Cellular deformation and partial and total lysis of cell components were seen in Staphylococcus pasteuri treated with whey F2 fraction under Transmission Electron Microscopy (TEM).