This study was aimed to determine antioxidant, antibacterial and angiotensin converting enzyme I inhibition activities of protein hydrolysates. Ultrasound and microwave pretreatment was applied to the beef. Hydrolysates were obtained by enzymatic (alkalase) hydrolysis at different times (60 min, 90 min, 120 min). Results showed that protein hydrolysates exhibited high percentages of hydrolysis of protein hydrolysates ranging from 64.1 to 88.93%. Angiotensin converting enzyme I inhibition activity ranging from 45.5 to 86.79%. DPPH inhibition values ranging from 9.25 to 29.32% and ABTS values ranging from 6.35 to 14.43 mg TEAC/g. E.coli and S.aureus were used in antibacterial activity at concentrations of 5 log cfu/ml and 7 log cfu/ml. It has been found that hydrolysates showed inhibitory activity against the tested pathogens. The results were significantly (P < 0.01) affected by ultrasound and microwave. These results indicated that ultrasound and microwave processes markedly increased the bioactive properties of protein hydrolysates obtained from an enzymatic hydrolysis.