Volatile compounds of hedge mustard (Sysimbrium officinale) have been investigated for the first time. Forthy-two compounds were identified after hydrodistillation (without or upon autolysis) after gas chromatography and gas chromatography/mass spectrometry analyses. In addition, after decoction and hydrolysis of O-glycosides, 18 volatile O-aglycones were identified. In general, the main volatiles found in hydrodistillates were: isopropyl isothiocyanate (27.6-48.9%), 2-methylpropanenitrile (0.5-18.8%), (Z)-hex-3-en-1-ol (0.5-18.0%), sec-butyl isothiocyanate (4.9-9.4%), (E)-hex-2-enal (3.5-8.6%), (Z)-hex-2-en-1-ol (0.3-8.4%), octanoic (0.5-8.6%) and dodecanoic acid (0-5.0%), 2-methylbutanenitrile (0-4.6%), dibutyl phthalate (0-4.5%), and ethyl linolenate (0-3.6%). The main volatile O-aglycones were: 2-phenylethyl alcohol (21.5%), 6,7-dehydro-7,8-dihydro-3-oxo-alpha-ionol (9.3%), eugenol (8.3%), benzyl alcohol (7.0%), ethyl vanillate (5.2%), 6-(tert-butyl)-5-methylphenol (5.1%), vanillin acetone (4.7%), ethyl 4-hydroxybenzoate (4.3%), and 2-hydroxy-beta-ionone (3.8%). All hydrodistillates exhibited great potential of antibacterial activity against five Gram-positive bacteria, nine ampicillin-resistant Gram-negative bacteria, and four fungi at a concentration of 500 microg/ml using the disc diffusion method.