This project investigates the role of extracellular membrane vesicles (MVs) in Staphylococcus aureus biofilm formation. To elucidate how MVs contribute to this process, we conducted a proteomic analysis to identify and compare proteins present in the S. aureus MN8 biofilm matrix, as well as proteins associated with MVs purified from MN8 biofilm and planktonic cultures. We identified 747 proteins in the biofilm matrix and 730 proteins in MVs purified from the biofilm, of which 576 were shared. Abundance-based correlation analysis revealed a strong positive correlation between these two protein sets. In contrast, only 568 proteins were detected in MVs derived from MN8 planktonic cultures, which exhibited a distinct composition, particularly in the cytoplasmic protein category, compared to biofilm-associated MVs. Overall, our findings demonstrate that the protein cargo of biofilm-derived MVs closely mirrors the biofilm matrix proteome, suggesting that MVs play a significant role in biofilm formation.