Background: Staphylococcus aureus is a clinically important pathogen. A small number of whole-cell fluorescence in situ hybridization (FISH) probes have been reported to detect S. aureus. New online computational tools for in silico design and testing make it possible to assess candidate FISH probes for S. aureus. Materials and Methods: Six online tools, NCBI-Nucleotide, Ribosomal Database Project, NCBI-Blast, Reverse-Complement, Probecheck, and mathFISH, were employed in a workflow to evaluate FISH probes for S. aureus. A previously reported probe, Staaur-16S69, was compared to a new probe, KT18-16S68, predicted by mathFISH to have the same performance. Results: A number of new probes for S. aureus were predicted to perform as well or better in silico as those previously reported. When tested in a FISH assay, Staaur and a new probe, KT18, were found to have the same performance. Conclusion: Existing and new FISH probes for S. aureus were found to be accurately identified and characterized with online computational tools. In silico evaluation of probes has the potential to reduce the time spent evaluating probes in the laboratory.