Hospital-acquired infections caused by enterococci have increased dramatically since the 1970s. Many nosocomial enterococcal bloodstream infections are associated with medical devices such as central venous catheters. The ability to form biofilm on medical devices is a potential virulence trait that may allow enterococci to cause infections in the expanding population of patients managed with such devices. In this study, the hypothesis that increased ability to form biofilm in vitro is associated with medical-device-related infection in vivo was tested. A microplate assay was employed to assess biofilm-forming characteristics of enterococci in 0.9 % (w/v) sodium chloride, an oligotrophic environment, and BHI, a nutrient-rich environment. Results were compared in isolates from different sources of infection. One hundred and nine enterococcal bloodstream isolates were assayed. Biofilm formation on microplates was demonstrated by all Enterococcus faecalis isolates and 16/38 (42 %) Enterococcus faecium isolates. E. faecalis isolates produced significantly more biofilm than E. faecium isolates in both media (P < 0.0001, Mann-Whitney U test). E. faecalis isolates from intravascular-catheter-related bloodstream infections (CRBSIs) produced significantly more biofilm than non-CRBSI isolates (P < 0.0001), or isolates of uncertain clinical significance (P < 0.0001). Biofilm formed by E. faecium isolates was not significantly affected by culture medium and did not differ between isolates from the different clinical categories. In conclusion, there was significantly more biofilm formed by E. faecalis isolates causing CRBSI compared with isolates from other types of infection or from isolates of uncertain clinical significance. The ability of E. faecalis isolates to form biofilm in vitro appears to be a marker of a virulence trait that enhances the ability of isolates to cause CRBSI.