Enterococcus faecalis is commonly isolated from different wound types. However, despite its prevalence, the pathogenic mechanisms of E. faecalis in wound infections remains poorly understood. We adopted an in vivo E. faecalis transposon sequencing approach to identify fitness factors that were crucial for initial colonization and persistence of E. faecalis during wound infections in a mouse model. We demonstrated that purine biosynthetic genes and a phosphotransferase system (PTS) were important for E. faecalis colonization and persistence, respectively. Purine metabolite levels were found to be lower in the wound site during initial colonization compared to the persistence phase, highlighting the importance of de novo purine biosynthesis during initial colonization. Furthermore, the PTS identified was characterized to be involved in the transport of mannose and galactose. Preliminary carbohydrate quantifications of the wound site using ELISA showed that mannose was higher during the persistence phase of infection compared to initial colonization, suggesting availability of various carbohydrates changes as the infection progresses, potentially impacting E. faecalis wound pathogenesis. Future work includes quantification of galactose and glucose (the main energy source) of E. faecalis-infected wound site to understand the carbohydrate profile as infection progresses. Also, to gain insight into how a mannose/galactose PTS contributes to the reduced fitness observed during the persistence phase, we performed an in vitro transcriptomic analysis using the mannose/galactose PTS gene deletion mutant (∆OG1RF_10021). When mannose was the sole carbohydrate source, shikimate and purine biosynthetic genes in the ∆OG1RF_10021 mutant were downregulated compared to the isogenic wild-type strain, indicating that mannose/galactose transport is interconnected with shikimate and purine biosynthesis. Together, our results demonstrate that dynamic microenvironment changes at the wound site affects pathogenic requirements and mechanisms of E. faecalis during infection and raise the possibility of inhibiting E. faecalis mannose/galactose transport to control wound infections.