In this study, Biarum bovei extract was used to produce bioactive peptides from wheat gluten protein and the biological and functional properties of the hydrolysates were determinated. The results showed that Biarum bovei extract has its highest protease activity (7.3 U/mg protein) at 45 °C and pH 5. Based on electrophoresis analysis, the molecular weight of hydrolysate was < 10 kDa. F1 fraction had the highest antioxidant activity in DPPH (65.85 ± 2.64 µmol TE/g)) and ABTS radical scavenging assays (295.81 µmol TE/g). F2 fraction with 86.3 ± 0.48 had the ability to inhibit the ACE enzyme. The F3 and F1 fractions had statistically the highest inhibition rate (49.37 ± 0.12%. and 79.19 ± 1.13%) in alpha-glucosidase and alpha amylase, respectively. The F1, F2 fractions hydrolysate had an inhibitory effect on Escherichia coli , Staphylococcus aureus , Listeria monocytogenes and Bacillus cereus . Functional properties of hydrolysates with increasing molecular weight, increased significantly. The presence of high levels (p ≤ 0.05) of amino acids with hydroxyl groups, hydrophobic and positive charged in fractions had critical role on biological and technological activity. These findings confirmed the efficiency of gluten hydrolysates with low molecular weight (F1 < 3 kDa) on biofunctionality such as scavenging radical activity, ACE inhibitory, antidiabetic and antibacterial activity could be beneficial from health and technological perspectives.