Chronic, non-healing wounds are a leading cause of prolonged patient morbidity and mortality due to biofilm- associated, polymicrobial infections. Staphylococcus aureus and Pseudomonas aeruginosa are the most frequently co-isolated pathogens from chronic wound infections. Competitive interactions between these pathogens contribute to enhanced virulence, persistence, and antimicrobial tolerance. P. aeruginosa utilizes the extracellular proteases LasB, LasA, and AprA to degrade S. aureus surface structures, disrupt cellular physiology, and induce cell lysis, gaining a competitive advantage during co-infection. S. aureus evades P. aeruginosa by employing aggregation mechanisms to form biofilms. The cell wall protein SasG is implicated in S. aureus biofilm formation by facilitating intercellular aggregation upon cleavage by an extracellular protease. We have previously shown that proteolysis by a host protease can induce aggregation. In this study, we report that P. aeruginosa proteases LasA, LasB, and AprA cleave SasG to induce S. aureus aggregation. We demonstrate that SasG contributes to S. aureus biofilm formation in response to interactions with P. aeruginosa proteases by quantifying aggregation, SasG degradation, and proteolytic kinetics. Additionally, we assess the role of SasG in influencing S. aureus biofilm architecture during co-infection in vivo, chronic wound co-infections. This work provides further knowledge of some of the principal interactions that contribute to S. aureus persistence within chronic wounds co-infected with P. aeruginosa, and their impact on healing and infection outcomes.