Enterococci are considered among the most prevalent global multidrug-resistant microorganisms globally. Their dissemination is a global concern, particularly by food-producing animals for both animals and humans. The aim of this study was to identify the species and investigate the antibiotic resistance and virulence profile of Enterococcus in bovine colostrum. Out of 88 presumptive Enterococcus isolates, species identification and susceptibility to 14 antimicrobials were tested using the disk diffusion method. An analysis of the antibiotic resistance and virulence genes was performed on the most prevalent species, using specific PCR assays. Enterococcus faecalis (54.5%), E. faecium (14.8%) and E. gallinarum (6.8%) were the identified species. To the best of our knowledge, this is the first report of E. gallinarum in bovine colostrum. The majority of the isolates showed resistance to quinupristin-dalfopristin (95.9%), erythromycin (80.7%), tetracycline (80.7%) and streptomycin (58%). Ninety-two percent of isolates were classified as multidrug-resistant. The most frequently detected resistance genes were tet(K) (61.1%), tet(M) (75.9%), tet(L) (90.7%), erm(B) (55.6%) and ant(6)-Ia (46.3%). The most prevalent virulence factors were cpd, esp, agg and cylLL. Enterococcus faecium showed a higher probability of carrying the erm(C), tet(M), ace and gel(E) genes (p < 0.05). These results demonstrated that colostrum can constitute an important reservoir and vehicle for the dissemination of antibiotic resistance and virulence genes to the three niches included in a One Health perspective (humans, animals and the environment), highlighting the importance of hygiene sanitary measures to mitigate colostrum microbial contamination.