Enterococci are ubiquitous microorganisms, indicators of faecal contamination and a part of normal intestinal microbiota, which are also capable of causing serious infections in humans and animals. They are also an important cause of nosocomial infections. The aim of the present study was to collect data about the prevalence of antimicrobial resistance and virulence genes in enterococci isolated from pet animals, livestock and food of animal origin in order to compare them with enterococci isolated from human clinical samples. A total of 854 Enterococcus isolates belonging to 16 species were analysed. Broth microdilution method was used to determine the minimum inhibitory concentrations of 12 antimicrobials. Polymerase chain reaction was used to detect the presence of seven virulence-associated genes (ace, asa1, cylA, efaA, esp, gelE and hyl). The highest resistance rate was found for tetracycline, erythromycin and ciprofloxacin. The susceptibility of enterococci for the critically important antimicrobials for human medicine was generally well conserved. The highest resistance rate was detected in isolates from human and animal clinical samples, broiler meat and poultry slaughterhouse, whereas the smallest proportion of resistance was observed in isolates from beef. In animals, 10 vancomycin-resistant isolates and 59 isolates with high-level aminoglycoside resistance were discovered. The highest occurrence of virulence-associated genes was observed in Enterococcus faecalis isolates, most commonly genes efaA, gelE and ace. Enterococcus faecium isolates most frequently harboured genes esp and hyl. In total, 28 distinct virulence types were identified. According to the previously detected genes encoding surface adhesins (ace, asa1 and esp), 92 isolates were selected to determine their biofilm forming ability on microtiter plates. The presence of all three aforementioned genes was significantly associated with their biofilm forming ability, as well as the sole presence of ace or esp gene, respectively, whereas no significant association was identified for asa1 gene. The results of the present study indicate a risk for direct transmission of resistant or highly virulent enterococci from animals to humans. In addition, enterococci represent an important reservoir of resistance and virulence-associated genes. The study is an important contribution to risk assessment regarding the transmission of resistant and virulent enterococci from animals to humans, either via food chain or pet animals.