Staphylococcus aureus is one of the major bacterial species that may cause clinical infection and food-poisoning cases. Strains of this bacterial species may produce a series of superantigens (SAgs) (i.e., staphylococcal enterotoxins [SEs], staphylococcal enterotoxin-like toxins, and toxic shock syndrome toxin). In this study, S. aureus strains from clinical samples and food-poisoning cases in Taiwan were collected; their SAg profiles, and SmaI digestion patterns determined by pulsed-field gel electrophoresis (PFGE), were then analyzed. Results showed that their SAg gene profiles and SmaI digestion patterns of chromosomal DNA were highly diverse. Although PFGE has been used as a criterion standard for typing of S. aureus strains, and the SAg profiles have been used in combination with PFGE for typing of S. aureus strains, we found that strains grouped in these combined patterns could be further discriminated by the random amplified polymorphic DNA (RAPD) method. Thus, the combined use of SAg profiles, PFGE, and RAPD patterns permits high discrimination for typing of S. aureus strains from not only the clinical samples but also the food-poisoning cases. Such a combined method may be used as a highly accurate approach for epidemiological study and tracing of the contamination origin of staphylococcal infections either in hospitals or food-poisoning cases.