In the present study, immunoglobulin Y (IgY) antibodies were raised in hens against the surface staphylococcal protein A (SpA) ofStaphylococcus aureus. Anti-SpA IgY were tested in vitro for diagnostic applications, bacteriostatic, and biofilm inhibition effects. A specific and sensitive immunocapture PCR (IPCR) was developed to detectS. aureusfrom food, clinical, and environmental samples. Anti-SpA IgY were used for capturingS. aureuscells from different matrices. Chicken antibodies were chosen over mammalian antibodies based on its inertness to immunoglobulin (Ig)-binding property of SpA protein. No cross-reactivity was encountered with closely related Gram-positive and Gram-negative food pathogens. Inter-assay variation is < 10%. The assay was found suitable for testing on solid and liquid food samples, skin, and nasal swabs. The assay showed limit of detection of >= 10(2) CFU/mL from broth cultures and 10(2) to 10(3) CFU/ml from diverse natural samples. This assay overcomes the false positives commonly encountered while using mammalian immunoglobulins (IgG). Anti-SpA IgY antibodies were tested for their bacteriostatic effect on the growth ofS. aureus. IgY antibodies at a concentration of 150 mu g/ml inhibited the growth ofS. aureuscompletely indicating the potential of IgY antibodies in neutralization of infectious pathogens. Similarly, anti-SpA IgY at MIC50 concentration reduced biofilm formation by similar to 45%. In view of advantages offered by IgY antibodies for specific detection ofS. aureusin immunocapture PCR (IPCR) assay and in vitro neutralization potential ofS. aureus, we recommend using IgY over conventional IgG of mammals involvingS. aureusand its antigens.