Abstract Background Staphylococcus aureus immunodominant surface antigen B (IsaB) elicits an immune response during septicemia and is generally classified as a virulence factor, but its biological function remains completely undefined. In an attempt to identify staphylococcal RNA-binding proteins, we designed an RNA Affinity Chromatography assay and subsequently isolated IsaB. Results Western analysis indicated that IsaB was both secreted and cell-surface associated. Gel Shift analysis confirmed the RNA binding activity but revealed that IsaB bound to any nucleic acid without sequence specificity. IsaB exhibited the highest affinity for double-stranded DNA followed by single-stranded DNA and RNA. Because extracellular DNA has been shown to play a role in biofilm formation, we investigated the biofilm-forming capacity of an isogenic isaB deletion mutant but we found that IsaB did not contribute to biofilm formation under any conditions tested. Conclusion IsaB is an extracellular nucleic acid binding protein, with little to no sequence specificity, but its role in virulence remains unclear.