The objective of this study was to determine the antimicrobial activity of proteolytic, meat-tenderizing enzymes (papain and bromelain) against E. coli and L. monocytogenes at three different temperatures (5, 25 and 35°C). Two overnight cultures of E. coli JM109 and L. monocytogenes were separately suspended in 1% peptone water and exposed to a proteolytic enzyme (papain or bromelain) at three different temperatures. Bromelain concentrations (4 mg/ml) and (1 mg/ml) tested at 25°C against E. coli and L. monocytogenes, respectively, were the most effective concentrations tested reducing populations by 3.37 and 5.7 log CFU/ml after 48 h, respectively. Papain levels of (0.0625 mg/ml) and (0.5 mg/ml) were the most effective concentration tested at 25°C against E. coli and L. monocytogenes, respectively, reducing populations by 4.94 and 6.58log CFU/ml after 48h, respectively. Interestingly, the lower papain concentration tested (0.0625 mg/ml) was more effective than the higher concentration (0.5 mg/ml) against E. coli at all three temperatures. As expected, the temperature was directly related to enzyme efficacy against both E. coli and L. monocytogenes.