Staphylococcus aureus is a multi-host pathogen that can form biofilms, which promote survival during infection and resilience to anti-microbials. S. aureus is a leading cause of bovine mastitis with major economic and animal welfare consequences; contemporary mastitis strains have evolved via historical host-switch events from humans. Investigations into the evolutionary adaptation of S. aureus to the dairy niche, we have identified a novel milk-associated biofilm phenotype. The biofilm is produced by bovine strains in bovine milk on materials used in milking equipment including steel, silicone, and rubber. We have discovered that expression of the extracellular protease aureolysin is required for both enhanced growth in milk and production of the milk-associated biofilm phenotype. Gene deletion mutants deficient in production of known biofilm-associated factors including aureolysin, intercellular adhesin, and Sortase A have been constructed in multiple bovine S. aureus lineages. In vitro assays indicated that milk-associated biofilms are dependent on aureolysin expression but that sortase A and intercellular adhesin are dispensable. However, biofilms formed in laboratory media, are unaffected by aureolysin production and dependent on sortase A or intercellular adhesin. Scanning electron microscopy shows that the milk-associated biofilm matrix exhibits a net-like structure with adherent bacterial cells—this is consistent across S. aureus from different lineages. Furthermore, liquid chromatography-mass spectroscopy indicates that milk proteins, including distinct casein chains, dominate the biofilm. Ongoing confocal microscopy is further investigating the main biofilm structural constituents. Overall, these data provide new insights into a novel biofilm phenotype that may promote the transmission and pathogenesis of mastitis.