Listeriosis has the highest fatality and hospitalization rate among foodborne illnesses. Listeria monocytogenes causes listeriosis and is a difficult bacterium for ready-to-eat food processors to eliminate because of its ability to grow in the absence of oxygen and under refrigeration. Recently, milk and its proteins have gained recognition as the largest source of biologically active peptides, and, it stands reason that several antimicrobial peptides (AMP) can be released from casein as it is the most abundant milk protein. AMPs are commonly obtained by cutting the whole protein into peptide fragments using enzymes or by acidification. The objective of this study was to predict potential AMPs through computer aided tools, improve hydrolysate preparation, and determine trypsin and pepsin-casein hydrolysate antimicrobial activity in growth media and on frankfurters against two strains of Listeria monocytogenes (Scott A and 310) and Escherichia coli O157:H7 (Salami strain). The prediction study procedure was to identify the most common variants of primary peptide sequences. The sequences were analyzed for greatest possible enzyme cuts on the protein, peptide masses, isoelectric point, net charge and percent hydrophilic residues using online proteomics programs. The fragments were explored for AMP commonalities: fragment length of 3 to 50 amino acids, positive (cationic) net charge, and hydrophilic residues between 25 and 50%. This technique identified 16 potential AMPs which proved that it is possible to screen for AMPs. The method used to determine the trypsin-casein hydrolysate (TCH) and pepsin-casein hydrolysate (PCH) antimicrobial activity was to hydrolyze sodium caseinate with pepsin or trypsin. L. monocytogenes (strains Scott A and 310) were incubated in 0, 10, 20, and 40% PCH and 0 and 50% TCH concentrations over a 24 hour period. PCH suppressed growth of L. monocytogenes Scott A by 1.76 log CFU/mL and reduced initial populations of L. monocytogenes 310 and E. coli O157:H7 by 0.52 and 0.62 log CFU/ml, respectively. TCH had little or no effect on growth suppression of any of the three test organisms. The frankfurter study was conducted by spot inoculating frankfurters with L. monocytogenes Scott A and then dipping frankfurters into one of five treatments (deionized water, pH 2.7 buffer, pH 5.1 buffer, pH 2.7 PCH, and pH 5.1 PCH) for 30 seconds; inoculated frankfurters that were not dipped served as controls. Frankfurters were incubated at 32°C for seven days. The results showed that there was no significant difference (p>0.05) in antimicrobial effectiveness among the treatments and control. This study demonstrated that enzymatically derived casein hydrolysates somewhat inhibit growth of L. monocytogenes and E. coli O157:H7 in culture media, but were ineffective when applied to frankfurters. Casein hydrolysate solutions can be easily made in a processing facility for application in fluid systems such as an antimicrobial spray on beef carcasses and in milk, juice, sports drinks, soda, soups, and yogurt. It also could be used in solid systems such as frankfurters, cheese, ground beef, and processed or RTE foods.