Ovine mastitis is a significant welfare and financial issue in dairy sheep flocks. In milking ewes, the main causative agent of clinical mastitis is Staphylococcus aureus and of subclinical mastitis coagulase-negative staphylococci. Biofilm production is a virulence factor of these bacteria, which contributes to their increased pathogenicity. Objective of the present study was to evaluate under field conditions the efficacy of a novel vaccine, which induces antibodies against the poly-N-acetyl β-1,6 glucosamine exopolysaccharide (PNAG), the main component of the extracellular biofilm matrix of Staphylococcus. Consequently, vaccination avoids biofilm formation by these organisms.