Aims:  To assess the inhibitory activity on Gram‐positive and Gram‐negative bacteria of several species of enterococci recovered from a natural corn silage. Methods and Results:  The inhibitory activity of strains of Enterococcus faecalis (58), Enterococcus faecium (35), Enterococcus gallinarum (3) and Enterococcus casseliflavus (4) were studied employing indicator strains from various sources (clinical, food and ATCC). Enterococcus faecalis MR99, the only strain with inhibitory activity, inhibited other enterococci, Listeria spp., Staphylococcus aureus, Clostridium spp., Bacillus spp., Escherichia coli, Shigella sonnei and Shigella flexneri. The bacterium contained only one conjugative pheromone‐responsive plasmid. The partially chromatography‐purified MR99 enterocin (PPE) had a molecular weight of approx. 5000 Da and a pI of 6·2, was sensitive to proteolytic enzymes and could be extracted in benzene and butanol. It appeared stable to adjustment of pH 4·0, 5·0, 6·0, 7·0 and 8·0 and was resistant to heat. Inactivation was at 15 min at 121°C. Enterocin MR99 was bactericidal on strains of Listeria monocytogenes, Staph. aureus, and bovine mastitis agents, it was bacteriostatic on E. coli. Although enterocins MR99 and AS48 have inhibitory activity on Gram‐negative bacilli, PCR studies demonstrated a lack of relationship between them. Conclusions:  The active component had a protein nature, was resistant to heat and presented a wide inhibitory spectrum. Significance and Impact of the Study:  The biological properties of Ent. faecalis MR99 suggest that this strain merits further investigations so it can be applied in human and veterinary health programmes.

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