Staphylococcus xylosus, a coagulase-negative Staphylococcus, is frequently isolated from food products of animal origin and used as a starter culture in these products in which it contributes to their flavour, while Staphylococcus aureus, a coagulase-positive bacterium, causes foodborne intoxication and is implicated in a broad diversity of infections in medical sector, notably in nosocomial infections. S. xylosus and S. aureus are both capable of forming a biofilm and share the same ecological niches, thus we explored their interaction in biofilms with a view to limiting the risks associated with S. aureus. Cell-free supernatants of different strains of S. xylosus were able to inhibit the biofilm formation of S. aureus. The S. xylosus C2a strain released into the supernatant a molecule of molecular weight above 30kDa that is resistant to proteolytic enzymes and inhibits the formation of S. aureus MW2 biofilm, though the mechanism involved has yet to be elucidated. Furthermore, S. xylosus C2a modified the architecture of S. aureus MW2 in co-culture biofilm. Confocal laser scanning microscopy revealed that S. aureus formed a biofilm with a flat and compact structure while in co-culture with S. xylosus the two species formed large juxtaposed aggregates throughout the period of incubation. This architecture made the S. aureus biofilm more susceptible to detachment.