Strains of Enterococcus faecium CWBI-B1430 and Enterococcus mundtii CWBI-B1431, isolated from artisanal-produced Peruvian cheeses, offer a broad potential application as food biopreservatives for heat-processing treatments. The aim of this work was to optimize the production conditions and medium composition for the production of bacteriocin-like substances (BLIS). The kinetics of the BLIS production for both strains is a growth-associated process, and the maximum inhibitory activity for E. faecium CWBI-B1430 and E. mundtii CWBI-B1431 was achieved after 9 and 8 h of fermentation, respectively. Temperature, initial pH, oxygen level, and their interactions all influence BLIS production for both strains; this influence was specific for each strain. The bacteriocin gene expression in a 20.0-L fermentor confirms previous work carried out by fermentation in the flask; the first reports of expression revealed mundticin KS from E. faecium and enterocin P from E. mundtii. Both strains demonstrated antilisteria activity and were multi-producers of enterocin. Furthermore, the study with whey milk as an inexpensive source of fermentable carbohydrates indicated lower BLIS production than that obtained with M17LG broth. The short chain fatty acid (SCFA) analyses offer a new alternative use for both strains, as strains probiotic, for propionic acid production. © 2011 Springer-Verlag and the University of Milan.