Whey, a by-product of cheese processing, has important nutritional qualities, as it is a rich source of proteins balanced with amino acids. As such, it has a broad range of functional and biological properties that can be exploited biotechnologically for diverse applications. Several studies have demonstrated that the enzymatic hydrolysis of whey proteins releases peptides that can perform biological activities, depending on the precise enzymatic process applied and the specific protein used. The present study examines the antimicrobial activity obtained from hydrolysates of protein concentrates of bovine whey by means of the aspartyl protease Eap1. It demonstrates antimicrobial activity against the following human non-opportunistic (Salmonella sp.) and opportunistic pathogens (Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli and Candida albicans), and then compares the findings obtained to the action of two commercial enzymes: trypsin (TP) and chymotrypsin (CMTP). Results indicate that the antimicrobial activity of the hydrolysates produced with Eap1 was higher than that generated by TP and CMTP. Also, the study determined that the Eap1 products were the only ones that inhibited growth of the yeast C. albicans.