Staphylococcus aureus is a leading cause of both hospital and community-associated infections that can manifest in a wide range of diseases. These diseases range in severity from minor skin and soft tissue infections to life-threatening sepsis, endocarditis and meningitis. Of rising concern is the prevalence of antibiotic resistant S. aureus strains in the population, and the lack of new antibiotics being developed to treat them. A greater understanding of the ability of S. aureus to cause infection is crucial to better inform treatments and combat these antibiotic resistant superbugs. The ability of S. aureus to cause such diverse infections can be attributed to the arsenal of virulence factors produced by the bacterium that work to both evade the human immune system and assist in pathogenesis. Many of these virulence factors have redundant or overlapping functions, and consequently, vaccines and antibiotics designed to target one or a small number of virulence factors have had limited success in combatting infection. An alternative approach to targeting individual factors has been to identify global regulators or systems that affect the expression of numerous virulence factors or virulence factor activity in the cell. Peptidyl-prolyl cis/trans isomerases (PPIases) are a family of enzymes that have been shown to regulate virulence factor activity and assist in protein secretion in numerous bacteria. In addition, the deletion of certain PPIases has resulted in an attenuation of virulence, demonstrating their importance in infection. The objective of this dissertation was to characterize three PPIase proteins: PrsA, PpiB and trigger factor (TF) encoded by S. aureus and determine if they contribute to virulence factor production, regulation and disease.