Enterococci may improve the typical taste and flavor of fermented foods through their proteolytic and lipolytic activities. However, some enterococcal strains are recognized as nosocomial pathogens, which have virulence genes and resistance to certain antibiotics. Enteroccocci are also found in human milk microflora. The aim of this study was to investigate the potential virulence genes and antibiotic resistance characteristics of Enterococcus faecalis isolates from human milk and colostrum samples. In total, 23 Enterococcus faecalis strains were identified from human milk and colostrum samples. Antibiotic-resistant E. faecalis isolates were determined using the disk diffusion method. Vancomycin resistance genes (vanA, vanB) and some virulence genes (agg(2), gelE, efaAfm, ccf, cpd, cad, cylM, cylB, etc.) were investigated using polymerase chain reaction (PCR). All strains were sensitive to ampicillin, penicillin G, chloramphenicol, and vancomycin. None of the E. faecalis isolates contained vanA, vanB, or efaAfm genes. The results of this study indicated that there were no harmful enterococci strains in human milk and colostrum samples in terms of tested virulence factors and antibiotic resistance. Therefore, the E. faecalis isolates from human milk may have the potential to be considered as a functional culture for the food industry.