Endocarditis isolates of Enterococcus faecalis produced biofilm significantly more often than nonendocarditis isolates, and 39% of 79 versus 6% of 84 isolates produced strong biofilm (P < 0.0001). esp was not required, but its presence was associated with higher amounts of biofilm (P < 0.001). Mutants disrupted in dltA, efaA, ace, lsa, and six two-component regulatory systems were largely unaltered, while disruptions in epa (encoding enterococcal polysaccharide antigen), atn (encoding an autolysin), gelE (encoding gelatinase), and fsr (encoding the E. faecalis regulator) [corrected] resulted in fewer attached bacteria, as determined using phase-contrast microscopy, and less biofilm (P < 0.0001).