Attachment of bacterial pathogens to the niche tissue in the host is the first step in biofilm formation leading to colonization and establishment of infection in the host. While the most common method used for determining the potential role of a bacterial antigen in biofilm formation has been demonstration of loss of this property using specific knockout mutants, it is an expensive and a laborious procedure. This study describes an alternative immunological assay for identification of attachment antigens of Staphylococcus aureus, potentially important in the development of an effective vaccine against infections caused by this pathogen. The method is based upon the concept of inhibition of attachment of S. aureus to PEGs coated with virulence antigen-specific antibodies. Antibodies used for validation of this assay were specific for ClfA, FnBPA, SdrD, PNAG and α-toxin, accredited biofilm-associated antigens of S. aureus.