Natural preservatives, such as enterocins, have been the focus of several studies for use in the food industry. However, the commercial media used to obtain enterocins are still expensive, presenting a disadvantage for large-scale production. In this study was developed four formulations of culture media containing soybean flour for obtaining Enterococcus durans enterocins. The antilisterial activity of E. durans MF5 enterocins obtained in soybean and MRS media (control) was characterized, with Listeria monocytogenes CLIP2032 and L. innocua CLIP12612 as the bacterial strains. The growth of E. durans (CFU/mL) was significantly affected by the incubation time in the soybean and MRS media (p <0.05), but the composition of the media did not affect the cell development of the enterocin-producing microorganism. When evaluating the genes encoding enterocin synthesis, positive results were obtained for enterocin A, B, P, and X. When treated with proteolytic enzymes (α-chymotrypsin, protease, and proteinase-K), enterocin was inactivated, confirming its protein character. The antilisterial activity of the enterocins obtained in soybean media was up to 6,400 AU mL-1. Enterocins from soybean media M1 and M2 showed antibacterial activity at a concentration of 1 mg/mL after 6 h incubation. Thus, we show that culture media formulated with soybean flour are promising substrates for enterocin production that would allow the protocol to be expanded on a large scale.