Enterococcus faecalis is a leading causative agent of catheter-associated urinary tract infection (CAUTI), the most common hospital-acquired infection. Its ability to grow and form catheter biofilm is dependent upon host fibrinogen (Fg). Examined here are how bacterial and host proteases interact with Fg and contribute to virulence. Analysis of mutants affecting the two major secreted proteases of E. faecalis OG1RF (GelE, SprE) revealed that while the loss of either had no effect on virulence in a murine CAUTI model or for formation of Fg-dependent biofilm in urine, the loss of both resulted in CAUTI attenuation and defective biofilm formation. GelE-, but not SprE- mutants, lost the ability to degrade Fg in medium, while paradoxically, both could degrade Fg in urine. The finding that SprE was activated independently of GelE in urine by a host trypsin-like protease resolved this paradox. Treatment of catheter-implanted mice with inhibitors of both host-derived and bacterial-derived proteases dramatically reduced catheter-induced inflammation, significantly inhibited dissemination from bladder to kidney and revealed an essential role for a host cysteine protease in promoting pathogenesis. These data show that both bacterial and host proteases contribute to CAUTI, that host proteases promote dissemination and suggest new strategies for therapeutic intervention.