Enterococcus faecalis is one of the most significant bacterial pathogens associated with the first-week mortality of chickens. Here, the surface properties of bacterial cells and the selected virulence factors of E. faecalis strains isolated from the hearts of clinically healthy broiler chickens were studied. Investigations were carried out on live and autoclaved cells. E. faecalis (ATCC 29212) was used as a reference strain. The bacterial cells revealed different haemolytic activities. Their surface free energy was dominated by the hydrophobic component. The cell walls of the bird isolates showed slightly weaker acidic characteristics than those of E. faecalis (ATCC 29212). Moreover, the bacterial cells from the chicken hearts showed higher electrophoretic mobility and surface electrical charge than the reference strain, and consequently demonstrated a low ability to form biofilms.