OBJECTIVES: This study characterized the multiple biological activities of the natural compound tomatidine against Staphylococcus aureus. Notably, this work examined the antibacterial activity of tomatidine in combination with other antibiotics and the influence of this compound on the expression of virulence factors in S. aureus. METHODS: The effect of tomatidine on the susceptibility of S. aureus to several antibiotic classes was determined by a broth microdilution procedure and a chequerboard protocol to measure fractional inhibitory concentration indices and to reveal drug interactions. Time-kill experiments for aminoglycoside/tomatidine combinations were also performed. The haemolytic ability of several strains in the presence of tomatidine was measured on blood agar plates and the expression of virulence-associated genes in strain ATCC 29213 treated with tomatidine was monitored by quantitative PCR. RESULTS: Tomatidine specifically potentiated the inhibitory effect of aminoglycosides but not of other classes of drugs. This potentiating effect was observed against strains of different clinical origins (human blood, cystic fibrosis airways, osteomyelitis, skin tissues and bovine mastitis), including aminoglycoside-resistant bacteria possessing the aac(6')-aph(2″), ant(4')-Ia and aph(3')-IIIa genes. The killing kinetics for the combination of aminoglycosides with tomatidine revealed strong bactericidal activity. Although tomatidine did not possess growth-inhibitory activity of its own against prototypical S. aureus, it inhibited the haemolytic activity of several strains and, more specifically, blocked the expression of several genes normally influenced by the agr system. CONCLUSIONS: These results show that tomatidine is an aminoglycoside potentiator that also acts as an anti-virulence agent targeting both antibiotic-susceptible and antibiotic-resistant S. aureus.