Staphylococcus aureus is a leading cause of chronic biofilm infections. Hyaluronic acid (HA) is a large glycosaminoglycan abundant in mammalian tissues that has been shown to enhance biofilm formation in multiple Gram-positive pathogens. We observed that HA accumulated in an S. aureus biofilm infection using a murine implant-associated infection model and that HA levels increased in a mutant strain lacking hyaluronidase (HysA). S. aureus secretes HysA in order to cleave HA during infection. Through in vitro biofilm studies with HA, the hysA mutant was found to accumulate increased biofilm biomass compared to the wild type, and confocal microscopy showed that HA is incorporated into the biofilm matrix. Exogenous addition of purified HysA enzyme dispersed HA-containing biofilms, while catalytically inactive enzyme had no impact. Additionally, induction of hysA expression prevented biofilm formation and also dispersed an established biofilm in the presence of HA. These observations were corroborated in the implant model, where there was decreased dissemination from an hysA mutant biofilm infection compared to the S. aureus wild type. Histopathology demonstrated that infection with an hysA mutant caused significantly reduced distribution of tissue inflammation compared to wild-type infection. To extend these studies, the impact of HA and S. aureus HysA on biofilm-like aggregates found in joint infections was examined. We found that HA contributes to the formation of synovial fluid aggregates, and HysA can disrupt aggregate formation. Taken together, these studies demonstrate that HA is a relevant component of the S. aureus biofilm matrix and HysA is important for dissemination from a biofilm infection.