Samples of the intestinal content and carcasses of wood pigeons (Columba palumbus) were evaluated for enterococci with antimicrobial activity. Enterococcus faecium comprised the largest enterococcal species with antagonistic activity, followed by Enterococcusfaecalis and Enterococcus columbae. PCR amplification of genes coding bacteriocins and determination of their nucleotide sequence, and the use of specific antipeptide bacteriocin antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, permitted characterization of enterococci coding that described bacteriocins and their expression. The efaAfm determinant was the only virulence gene detected in E. faecium, whereas E. faecalis showed a larger number of virulence determinants, and E. columbae did not carry any of the virulence genes examined. Although all E. faecalis isolates manifested a potent direct antimicrobial activity, no activity was detected in supernatants of producer cells. Purification of the antagonistic activity of E. columbae PLCH2 showed multiple chromatographic fragments after matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analysis, suggesting the active peptide(s) had not yet purified to homogeneity. Bacteriocinogenic E. faecium and E. columbae isolates may be considered hygienic for production of enterocins and potentially safe due to their low incidence of potential virulence genes and susceptibility of most relevant clinical antibiotics. However, the presence among the enterococci of E. faecalis strains with a potent antagonistic activity and multiple virulence factors is an issue that must be considered further.