Staphylococcus aureus is a remarkably pathogenic bacterium that is widely prevalent among the human population. It is the leading agent of skin and soft tissue infections, and is also responsible for causing an array of severe and life threatening diseases. The invasiveness of the pathogen, coupled with increasing antibiotic resistance seen for S. aureus infections, makes this bacterium a prominent public health concern. The extended pathogenicity of S. aureus is largely due to its repertoire of virulence factors, which are typically characterized by being bound to the cell wall, or secreted into the extracellular environment. Previously, our lab identified a leucine aminopeptidase mutant (pepZ) as being strongly attenuated in virulence for both systemic and localized models of infection. Importantly, PepZ marked the first intracellular bacterial aminopeptidase found to be involved in pathogenesis in Gram-positive bacteria. In this study, we set out to explore the role of the remaining eleven, non-essential, uncharacterized aminopeptidase enzymes in S. aureus disease causation, and present two additional aminopeptidase genes, pepT1 and pepT2, which are important for virulence in both human ex vivo models, and murine in vivo models of disease. Interestingly, these enzymes do not appear to be necessary for the utilization of free peptides for cellular nutrition and metabolism, which is a typical characteristic of aminopeptidases. Transcriptional analysis reveals maximal expression of pepT1 and pepT2 during early exponential growth phase, while localization mapping demonstrates that the PepT1 and PepT2 enzymes are found in the bacterial cytoplasm during all stages of growth. To explore these findings on a global level, an in-depth proteomic investigation of cleavage properties and cellular substrates identified several proteins as having significant changes in N-terminal peptide abundance in pepT1 and pepT2 mutant proteomes. We identified a number of putative independent and shared targets for the PepT1 and PepT2 enzymes that are known to impact cellular fitness and pathogenesis, including: DnaK, a heat shock protein conserved throughout all organisms, which functions to help deal with mis-folded and aggregated proteins that have accumulated as a result of cellular stress; ArlR, the response regulator of the ArlRS two-component system, which is an important regulator of agglutination and virulence in S. aureus; and of special interest, ClpC, an ATP-dependent protease chaperone which is a component of the primary machinery by which protein degradation occurs in S. aureus. Collectively, our data proves the importance of the PepT aminopeptidase enzymes in S. aureus pathogenesis, and indicates these enzymes are likely involved in bacterial stress response and virulence by functioning through the bioactivation/inactivation of key cellular proteins.