Aims: The isolation and partial characterization of anti-Listeria bacteriocin producing strains present in milk from areas of northern Greece in view of their potential use as protective cultures in food fermentations. Methods and Results: Three hundred and thirty-two isolates were obtained from milk samples intended for Feta cheese production and gathered from 40 individual producers in Northern Greece. Isolates with anti-Listeria activity were identified by multiplex PCR as Enterococcus faecium and grouped by (GTG)(5)-PCR. The genomes of the anti-Listeria isolates were examined for the presence of known enterocin genes and major virulence genes by means of specific PCR. At least three known enterocin encoding genes were present in the genome of each of the 17 isolates. None of the 17 isolates harboured any of the virulence genes tested for or exhibited haemolytic activity. Conclusions: Enterococcus faecium was the dominant anti-Listeria species in the milk samples. The isolates had the potential of multiple bacteriocin production and did not exhibit some important elements of virulence. Significance and Impact of the Study: Enterococci present in milk of this area of northern Greece may be partly responsible for the safety of Feta cheese and could be useful for the production of anti-Listeria protective cultures.