Enterococcus mundtii Tw56 is a bacteriocin-producing strain that was isolated from intestinal content of silverside (Odontesthes sp.). The aim of the present work was to determine physicochemical factors and culture medium composition for higher yield of viable cells and bacteriocin production. No changes were observed in the antimicrobial production when glucose was replaced by fructose or maltose in the MRS medium formulation. On the other hand, highest bacteriocin activity was obtained when meat extract was used as a sole nitrogen source. While the maximun biomass was achieved at 35 oC, the optimal temperatures for bacteriocin production were observed at 25 and 30 oC. The optimal initial pH for cell growth and bioactivity was 6.5, both parameters dropped when the experience started at pH 6.0 or 5.5. Biomass formation and bacteriocin production decreased in the presence of sodium chloride. The strain started producing the bacteriocin at the late exponential phase. The activity increased as a function of the cell mass and reached the maximun at the end of exponential phase (12 h). A decrease of antimicrobial activity was observed in the stationary phase (16 h), possibly due to degradation by proteolitic enzimes.