Aims To determine the sensitivity of five strains of Staphylococcus aureus to five essential oils (EOs) and to investigate the anti-biofilm activity of lemongrass and grapefruit EOs. Methods and Results Antimicrobial susceptibility screening was carried out using the disk diffusion method. All of the strains tested were susceptible to lemongrass, grapefruit, bergamot and lime EOs with zones of inhibition varying from 2.85 to 8.60cm although they were resistant to lemon EO. Lemongrass EO inhibited biofilm formation at 0.125% (v/v) as measured by colorimetric assay and at 0.25% (v/v) no metabolic activity was observed as determined by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction. Grapefruit EO did not show any anti-biofilm activity. Following exposure to lemongrass EO extensive disruption to Staph.aureus biofilms was shown under scanning electron microscopy. Conclusions In comparison to the other EOs tested, lemongrass exhibited the most effective antimicrobial and anti-biofilm activity. Significance and Impact of the Study The effect of lemongrass EO highlights its potential against antibiotic resistant Staph.aureus in the healthcare environment.