Enterococcal surface protein (Esp) is a cell wall-attached virulence factor of the leading nosocomial pathogens Enterococcus faecalis and E. faecium and is found predominantly in disease-causing strains. The presence of esp is associated with increased bacterial burden in rodent models of enterococcal endocarditis and urinary tract infection. The mechanism by which Esp enhances colonization of tissues is unknown. The presence of Esp has also been found to enhance biofilms, as assayed by crystal violet staining, in some but not all strains of Enterococcus. The circumstances under and extent to which Esp enhances biofilms are not well understood, and conflicting data exist, leaving its role in biofilms undefined. To address the functional role of Esp, we sought to use X-ray crystallography to determine the structure of the unique N-terminal region of Esp, as structure is often indicative of function. We successfully determined the structure of the first 405 amino acids of Esp (Esp452), which revealed that this region is composed of two Ig-type domains typical of bacterial adhesins. While we did not detect binding to host factors, we did find that Esp452 strengthens enterococcal biofilms against mechanical and enzymatic challenge. Further examination revealed that this activity is correlated with the formation of amyloid-like fibrils in a pH-dependent manner. Interestingly, a larger fragment consisting of the entire N-terminal region of Esp, Esp743, did not demonstrate this activity, despite containing Esp452. We used a structural approach to explore the possibility of auto-inhibition, and found by small-angle X-ray scattering that the additional 291 amino acids contained in Esp743 appear to have close contacts with Esp452, supporting a role in structural stabilization of the protein and prevention of the formation of amyloid fibrils. Finally, we explored mechanisms of activation of the N-terminal region. We found that at pH 4.0, Esp743 was capable of strengthening biofilms, suggesting that extreme pH may be sufficient to generate activity of cell wall-attached protein. Alternatively, we found that a shorter truncation fragment of the N-terminal region, consistent with cleavage by a human protease, was also capable of strengthening enterococcal biofilms.