Austrian veterinary (n=91), dairy (n=86), and human strains (n=48) of Staphylococcus aureus were tested for various phenotypic properties including clumping factor, egg-yolk reaction, production of thermonuclease and susceptibility to 14 antibiotics. In addition the expression of enterotoxins (A–E), and the presence of enterotoxin genes sea to sej and tst was determined. Significant differences in antimicrobial susceptibility were found with 84·6% of veterinary, 57·0% of dairy, and 20·8% of human strains susceptible to all antibiotics tested (P<0·0005). More human strains produced enterotoxins (41·7%) than veterinary (9·9%) and dairy strains (12·6%) while 40·7% and 38·5% of veterinary, 47·7% and 52·3% of dairy, and 77·1% and 87·5% of human strains were se- and tst-positive, respectively. AFLP analysis revealed nine clusters with over- or under-representation of strains with specific characteristics. Strains clustered according to origin (veterinary, dairy, and human) and/or presence of toxin genes and antimicrobial resistance.