Aims: Characterization of Ent F- 58 produced by Enterococcus faecium strain F58 isolated from Jben, a soft, farmhouse goat's cheese manufactured without starter cultures. Methods and Results: E. faecium strain F58 was isolated because of its broad inhibitory spectrum, including activity against food- borne pathogenic and spoilage bacteria. The antimicrobial substance was produced during the growth phase, with maximum production after 16 - 20 h of incubation at 30 degrees C, and was stable over a wide pH range ( 4 - 8) and at high temperatures ( 5 min at 100 degrees C). The enterocin was purified to homogeneity using cation exchange and hydrophobic interaction on C- 18 and reverse- phase high- performance liquid chromatography. The activity was eluted as two individual active fractions ( F- 58A and F- 58B) and matrix- assisted laser desorption/ ionization time- of- flight mass spectrometry analysis showed masses of 5210.5 and 5234.3 Da respectively. Both peptides were partially sequenced by Edman degradation, and amino- acid sequencing revealed high similarity with enterocin L50 ( I). PCR- amplified fragments containing the structural genes for F- 58 A and B were located in a 22-kb plasmid harboured by this strain. We verified that it also holds the structural gene for P- like enterocin. Conclusion: E. faecium strain F58 from Jben cheese, a producer of enterocin L50, exerts an inhibitory effect against strains of genera such as Listeria, Staphylococcus, Clostridium, Brochothrix and Bacillus. Enterocin was characterized according to its functional and biological properties, purification to homogeneity and an analysis of its amino acid and genetic sequences. Significance and Impact of the Study: E. faecium strain F58 is a newly discovered producer of enterocin L50, the biotechnological characteristics of which indicate its potential for application as a protective agent against pathogens and spoilage bacteria in foods.