Sheep milk whey proteins are becoming more well-known for their bioactivity or health-promoting properties. Furthermore, the natural digestion of sheep whey proteins in the gastrointestinal tract generates peptides with a wide range of bioactivities, including antihypertensive, opioid, antibacterial, antioxidant, and immunomodulatory properties. In the current study, purified sheep whey protein has been applied by using size-exclusion chromatography (SEC), and it was hydrolyzed by pepsin and alcalase enzymes at concentration 2.5% (w/w) at different times 30, 60, 120 and 240 min. The highest active time hydrolysate was subsequently fractionated by SEC and monitored for its antibacterial and antioxidant activities and characterization using UHPLC-MS/MS. Alcalase enzyme showed a significantly (p˂0.05) higher degree of hydrolysis reached to 30% than pepsin enzyme. While pepsin fraction (P5) characterized by significantly (p˂0.05) higher antimicrobial activity reach to 3.18 and 2.24 log reduction against E.coli and S.aureus, respectively. For alcalase fraction (A9) exhibited significantly the highest antioxidant activity rescuing 100% of the yeast cell from Hydrogen peroxide induced oxidative stress. Proteomic analysis of the highly active fractions identified peptides from α-lactalbumin and β-Lactoglobulin from sheep whey protein with structural similarity to known antioxidant peptides and antimicrobial peptides respectively. Thus, current results supported the using food grade enzymes like alcalase and pepsin in the food industry to obtain bioactive peptides which acts as natural antimicrobial and antioxidant agent