Glucosinolates (GLs) were characterized in various aerial parts (stems, leaves, and flowers) of Aurinia leucadea (Guss.) C. Koch and quantified according to the ISO 9167-1 official method based on the HPLC analysis of desulfoglucosinolates. Eight GLs, i.e., glucoraphanin (GRA), glucoalyssin (GAL; 1), gluconapin (GNA; 2), glucocochlearin (GCC), glucobrassicanapin (GBN; 3), glucotropaeolin (GTL), glucoerucin (GER), and glucoberteroin (GBE) were identified. The total GL contents were 57.1, 37.8, and 81.3 μmol/g dry weight in the stems, leaves, and flowers, respectively. The major GL detected in all parts of the plant was 2, followed by 1 and 3. GC/MS Analysis of the volatile fractions extracted from the aerial parts of fresh plant material either by hydrodistillation or CH(2) Cl(2) extraction showed that these fractions mostly contained isothiocyanates (ITCs). The main ITCs were but-3-enyl- (55.6-71.8%), pent-4-enyl- (7.6-15.3%), and 5-(methylsulfinyl)pentyl ITC (0-9.5%), originating from the corresponding GLs 2, 3, and 1, respectively. The antimicrobial activity of the volatile samples was investigated by determining inhibition zones with the disk-diffusion method and minimal inhibitory concentrations (MIC) with the microdilution method. They were found to inhibit a wide range of bacteria and fungi, with MIC values of 2.0-32.0 μg/ml, indicating their promising antimicrobial potential, especially against the fungi Candida albicans and Rhizopus stolonifer as well as against the clinically important pathogen Pseudomonas aeruginosa.