Staphylococcus aureus and Pseudomonas aeruginosa are bacterial pathogens that have been shown to co-exist in biofilms related to numerous infections. Although the interaction between these two species is competitive, both partially benefit from the coexistence. In this study, we exhaustively characterized the interaction between Staphylococcus aureus and Pseudomonas aeruginosa by utilizing a proteomics approach, individually targeting the surface-associated proteins (surfaceome), and proteins secreted or otherwise liberated to the extracellular space (exoproteome). To that end, the conditions to co-culture S. aureus and P. aeruginosa in vitro were optimized and a high-resolution proteomics approach was applied to compare surface-associated and extracellular protein profiles between mono- and co-cultured biofilms.