The transition of bacteria from a planktonic lifestyle to a biofilm is influenced by environmental stimuli, including surface adhesion and fluid flow. However, the effects of these factors in gram-positive bacteria are not well-understood. Enterococcus faecalis, which accounts for 5-15% of infective endocarditis (IE) cases, encounters pulsatile blood flow and high shear stress on heart valves. To investigate how fluid flow impacts E. faecalis virulence in IE, we used microfluidics to simulate heart valve shear stress and exposed surface-adhered bacteria to pulsatile fluid flow. After 30 minutes, transcriptional analysis revealed significant downregulation of the Fsr quorum sensing (QS) system suggesting that fluid flow interferes with autoinducer accumulation and QS activation during early IE. Consistent with this, E. faecalis fsr mutants are able to establish IE as well as the parental wild type strain in a rat model, indicating a limited role for QS in early infection. Surprisingly, fsr deletion mutants developed significantly larger cardiac vegetations at later stages of infection, characterized by extensive biofilm coverage and larger biofilm microcolonies resistant to neutrophil infiltration and antibiotic clearance. Clinically, 2/3 of E. faecalis isolates from IE patients contain inactivating deletions in the fsr locus, which correlates with increased disease severity. One explanation for the increased bacterial burden of fsr mutants is the role of gelatinase, a protease that is positively regulated by the Fsr QS system. We found that gelatinase can activate a human pro-inflammatory cytokine, suggesting that QS inactivation – and the resulting reduction of gelatinase - promotes an immunosuppressive microenvironment, enhancing biofilm development and persistence. These findings offer insight into the prevalence of QS mutations in clinical E. faecalis isolates, suggesting that while QS may facilitate biofilm formation in certain environments, its loss may confer a selective advantage during mammalian infection.