The aim of this study was first to ascertain the chemical composition and the physicochemical properties of cereal extracted β-glucan from barley flour. Secondly, to assess the antioxidant properties and the antibacterial properties of extracted β-glucan hydrolysates. The proximate composition, FT-IR and scanning electron microscopy of extracted β-Glucan were studied. Hydrolysates from extracted β-glucan, obtained by lichenase EGL from Penicillium occitanis, were a mixed linkage beta-oligosaccharides (MLBO) of trisaccharides and tetrasaccharides. MLBO showed a DPPH radical scavenger with IC50 about 1.8 ± 0.01 mg/mL whereas the IC50 of extracted β-glucan was about 5 ± 0.01 mg/mL. MLBO showed a high antioxidative capacity (175 μmol/mL α-tocopherol equivalents) at 5 mg/mL. The antimicrobial activity was confirmed against all tested bacteria especially at 20 mg/mL of MLBO while no inhibition was observed for all the strains used after the addition of either EGL or extracted β-glucan.