Recently identified innovative food preservatives, enterocins, bacteriocins produced by enterococci, have promising applications because most of them are active towards food pathogens. The biochemical characterization of an enterococcal strain, isolated from salami manufacturing, showed that it belongs to Enterococcus casseliflavus/gallinarum species. Furthermore, it produces a bacteriocin with a strong activity towards Listeria monocytogenes. For this reason, this LAB may be applicable in order to reduce the risk on Listeria outgrowth in food. Bacteriocin production kinetics is strictly dependent on the cultural conditions. So, the carbon and nitrogen sources of a growth medium suitable for production were optimised. Batch fermentations allowed to assess that bacteriocin production and release was predominant in the exponential phase, following a typical primary metabolite production kinetics. Contemporary, the lactic acid production from glucose was characterized, showing a homolactic fermentation. Due to growth-related production, different fed-batch strategies were implemented to improve the biomass and bacteriocin production. Interestingly, a suitable feed profile, based on the glucose consumption, and switched on at the end of the batch exponential phase, allowed to improve about 8-fold both the biomass concentration and the bacteriocin activity in the medium. Finally, the product activity was tested towards different microorganisms.