The proteome is defined as the set of proteins that an organism can produce. By extrapolation, the “Proteome Phenotype” corresponds to the set of proteins whose synthesis is modified (under- or over-expressed) in a genetically altered cell. This approach has been successfully used to identify co-regulated genes in some Gram positive and Gram negative bacteria but can also be carried out with cells mutated in non regulatory genes in parallel to physiological phenotype studies. Over past ten years, such results have been obtained with several mutant strains of Enterococcus faecalis, a rogue commensal-turned-pathogen and Gram positive bacteria. Systematic 2-D gel electrophoresis carried out with cells affected in genes encoding stress proteins or transcriptional regulators such as CcpA, HypR, or TCSs (Two Component Systems) allowed to characterize some members of these regulons and to identify pleiotrophic effects of some mutations. Moreover, comparison of gels obtained with proteins extracted from cells harvested after exposure to different stress conditions revealed “Stress Proteome Signatures” of E. faecalis. The availability of the E. faecalis proteome, updated in this review, is the key database to utilize these results for biomedical applications. Indeed, 46 proteins are now characterized on a master gel which allowed identification of some effectors of the stress response. Because E. faecalis is a member of the gastrointestinal tract flora, such proteomics approach may help to understand how it is able to cope with different hostile environments and then become a pathogen responsible for infections in the bloodstream, urinary tract, or surgical wound infections.