The purpose of this study was to investigate the inhibitory/bactericidal activity and cell membrane effects of the hydrodistilled essential oil of Inula helenium L. roots against Staphylococcus aureus. Additionally, detailed chemical investigation was done in order to pinpoint the most active oil constituents and also the parts of these molecules responsible for their antimicrobial effect. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the broth microdilution method. The membrane-active nature of this oil was investigated by measuring the culture turbidity, leakage of phosphates, and 260-nm-absorbing material, together with lysis of the exposed cells. Finally, the effect of the oil on the cells was visualized using scanning electron microscopy (SEM). The chemical composition of the essential oil was analyzed using gas chromatography-mass spectrometry (GC-MS) and preparative medium-pressure liquid chromatography (MPLC). Chemical modification of the oil was performed using catalytic hydrogenation (H(2), Pd/C) and reduction with NaBH(4). The MIC and MBC values were 0.01 μl mL(-1) and 0.02 μl mL(-1), respectively. Membrane damage was demonstrated through increased permeability (phosphates and nucleic acid leakage), followed by lysis of the exposed cells, captured on SEM images. The most active constituents were alantolactone, isoalantolactone, and diplophyllin. The essential oil showed very potent antistaphylococcal activity, with obvious membrane-damaging effects. Sesquiterpene lactones were found to be the most active principles of the oil, whose eudesmane core olefinic bonds, along with the α,β-methylene-lactone ring, are essential structural parts responsible for the exhibited antimicrobial activity.