Enterococcus faecalis is commonly isolated from different wound types. However, despite its prevalence, the pathogenic mechanisms of E. faecalis during wound infections remain poorly understood. We adopted an in vivo E. faecalis transposon sequencing and RNA sequencing approach to identify fitness determinants that are crucial for early colonization and persistence of E. faecalis during wound infections in a mouse model. We demonstrated that E. faecalis purine biosynthesis genes are important, likely because purine metabolites are low in wounds during early wound colonization. Furthermore, we found that the E. faecalis MptABCD phosphotransferase system (PTS) involved in the uptake of galactose and mannose, is crucial for E. faecalis persistence in wounds; and that carbohydrate availability changes as the infection progresses. To understand how MptABCD PTS contributes to the reduced fitness observed during E. faecalis persistence in wounds, we performed an in vitro transcriptomic analysis using the galactose/mannose PTS gene deletion mutant (∆mptD). When mannose was the sole carbohydrate source, shikimate and purine biosynthesis genes in the ∆mptD mutant were downregulated compared to the isogenic wild-type strain, indicating that mannose transport is interconnected with shikimate and purine biosynthesis. Together, our results suggest that dynamic and temporal microenvironment changes at the wound site affect pathogenic requirements and mechanisms of E. faecalis during infections and raise the possibility of inhibiting purine biosynthesis and/or MptABCD PTS to control wound infections.