Background: As a proof-of-concept, the feasibility of detecting Staphylococcus aureus faster than previous whole-cell fluorescent in situ hybridization (FISH) methods was tested. Methods: Isolates of Staphylococcus were treated with three rapid slide-based FISH protocols and DNA probes. Protocols were shortened by optimizing, combining or omitting steps. Results: All FISH protocols detected S. aureus and not the phenotypically similar Staphylococcus epidermidis. The express FISH assay was completed in 24 minutes. The one-step FISH assay with NaCl and the one-step with phosphate buffered saline (PBS) assay took 19 minutes, but yielded a weaker signal. Conclusions: The exploratory study identified S. aureus two to three times faster than previous methods. Additional testing in a clinical laboratory scenario (for example with positive blood-culture bottles) is warranted.