In recent years, the demand for innocuous and natural antimicrobials in food preservation has been increasing, resulting in research into novel antimicrobial agents as antimicrobial peptides. Argentine croaker (Umbrina canosai) is a fish species underutilized and has low commercial value, thus attracts the interest to obtain high added-value products, such as protein hydrolysates. The objectives of this study were to obtain protein hydrolysates from Argentine croaker muscle proteins by pH shifting process and with different degrees of hydrolysis (DH: 10 – 20%) using a commercial protease and to evaluate the antimicrobial activity of the different hydrolysates obtained. The proteins were recovered from Argentine croaker muscle by alkaline solubilization at pH 11.2 with 1 M NaOH and isoelectric precipitation at pH 5.0 with 1 M HCl. Hydrolysates were obtained by enzymatic hydrolysis of protein isolate using the enzyme Alcalase at optimum hydrolysis condition used (pH 8.0 and 50 °C). The DH of proteins by the pH-stat method at achieve a 10 and 20% DH was determined. Antimicrobial activity of these hydrolysates was tested by disk diffusion test (hale measured in cm) against different Gram-positive and Gram- negative microorganisms such as Escherichia coli, Shigella sonnei, Vibrio parahaemolyticus, Brochothrix thermosphacta, Listeria innocua and Staphylococcus aureus. Results showed that an increase in the DH resulted in higher content of hydrophobic and decrease in molecular weight (MW) distribution for hydrolysates obtained. Only Gram-positive microorganisms were inhibited. The B. thermosphacta, S. aureus and L. innocua showed relevant inhibition achieved of 1.11 cm, 1.20 cm and 0.98 cm by hydrolysate with DH 20%, respectively. Finally, it was possible to affirm that this effective antibacterial hydrolysate could be used to control undesirable bacteria and improve the safety in food.