ABSTRACT Rapid diagnosis is essential for the management of Staphylococcus aureus infections. A host recognition protein from S. aureus bacteriophage phiSLT was recombinantly produced and used to coat streptavidin latex beads to develop a latex agglutination test (LAT). The diagnostic accuracy of this bacteriophage-based test was compared with that of a conventional LAT, Pastorex Staph-Plus, by investigating a clinical collection of 86 S. aureus isolates and 128 coagulase-negative staphylococci (CoNS) from deep tissue infections. All of the clinical S. aureus isolates were correctly identified by the bacteriophage-based test. While most of the CoNS were correctly identified as non- S. aureus isolates, 7.9% of the CoNS caused agglutination. Thus, the sensitivity of the bacteriophage-based LAT for identification of S. aureus among clinical isolates was 100%, its specificity was 92.1%, its positive predictive value (PPV) was 89.6%, and its negative predictive value (NPV) was 100%. The sensitivity, specificity, PPV, and NPV of the Pastorex LAT for the identification of S. aureus were 100%, 99.2%, 98.9%, and 100%, respectively. Among the additionally tested 35 S. aureus and 91 non- S. aureus staphylococcal reference and type strains, 1 isolate was false negative by each system; 13 and 8 isolates were false positive by the bacteriophage-based and Pastorex LATs, respectively. The ability of the phiSLT protein to detect S. aureus was dependent on the presence of wall teichoic acid (WTA) and corresponded to the production of ribitol phosphate WTA, which is found in most S. aureus clones but only a small minority of CoNS. Bacteriophage-based LAT identification is a promising strategy for rapid pathogen identification. Finding more specific bacteriophage proteins would increase the specificity of this novel diagnostic approach.