Enterococcus faecalis is a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades. E. faecalis must be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-based in vivo expression technology (RIVET) to identify promoters on the E. faecalis OG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putative in vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected in in vitro- and in vivo-grown cells, providing the first evidence of in vivo-expressed antisense RNAs in E. faecalis. Deletions in the in vivo-activated genes that encode glutamate 5-kinase (proB [EF0038]), the transcriptional regulator EbrA (ebrA [EF1809]), and the membrane metalloprotease Eep (eep [EF2380]) did not hinder biofilm formation in in vitro assays. In a rabbit model of endocarditis, the ΔebrA strain was fully virulent, the ΔproB strain was slightly attenuated, and the Δeep strain was severely attenuated. The Δeep virulence defect could be complemented by the expression of the wild-type gene in trans. Microscopic analysis of early Δeep biofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.