Staphylococcus aureus is a major pathogen involved in ruminant mastitis. Staphylococcal mastitis is remarkably variable in terms of severity ranging from subclinical to gangrenous manifestations. It is also prone to recurrence partly because of S. aureus ability to invade Mammary Epithelial Cells (MEC). We investigate what makes a strain more or less virulent by identifying staphylococcal factors involved in mastitis acuteness. We also take account of ecological factors (udder ecosystem) that may modulate S. aureus virulence expression. S. aureus strains, isolated from gangrenous (O11) or subclinical (O46) ewe mastitis, were selected for identifying potential factors involved in mastitis acuteness. Despite a close phylogenetic relationship, these two strains reproducibly induce severe (O11) or mild (O46) symptoms in experimental ewe mastitis. Using a comprehensive omic characterization (genome, transcriptome, proteome and seroproteome), we showed dramatic differences between O11 and O46 relying on their ability to adapt and express virulence in a mammary context. The exact role of the genes identified here (exoproteins, mobile genetic elements, iron metabolism), and likely involved in mastitis acuteness and pathogen-host interactions, remains to be determined. Besides, we investigated the inhibitory potential of the lactic acid bacterium, Lactobacillus casei, naturally present in the teat ecosystem, against invasion of MEC by ruminant S. aureus isolates. Our results indicated L. casei strains impaired, in a contact-dependent way, adhesion and internalization of S. aureus without altering viability of MEC. Altogether, these results open avenues for the development of prevention strategies against mastitis.