Previous studies have demonstrated that Gynandropsis gynandra emits acetonitrile as a foliar volatile from intact plants and isolated leaves, and that this compound is an effective spider mite repellent. This study has used gas chromatography–mass spectrometry to investigate volatile compounds emitted from homogenised G. gynandra leaves to evaluate their tissue acetonitrile content and to look for other compounds that might be exploited for the management of spider mites. Acetonitrile was absent from the homogenised tissues of five lines of G. gynandra, studied over two seasons. Thirteen volatile compounds were emitted by G. gynandra at significantly higher levels than mite-susceptible pot roses, including isothiocyanates, aldehydes, esters, alcohols and terpenes. Six representative compounds were selected to assess bioactivity. Spider mite populations were completely inactive after a 2¿h exposure to butyl isothiocyanate, 2,4-heptadienal or ß-cyclocitral, when evaporated from 0.5¿µL of pure compound in a 100¿mL air space. The same level of inactivity was achieved after exposure to 5.0¿µL of (Z)-2-pentenol or a 25¿µL volume of 50% v/v Z-3-hexenal or 5% w/v methyl isothiocyanate. Dissipation of ß-cyclocitral following 24 h exposure to its concentration of 5¿µL in a 100¿mL air space resulted in a 6% recovery of the spider mites but at higher concentrations no recovery was observed. These identified compounds may have potential as extracted products for management of spider mites in roses, and a high constitutive content of them in roses may be of value in targeted plant breeding for enhanced insect resistance. The range of isothiocyanates found in G. gynandra accounts for the bitter taste of the leaves when used as a traditional vegetable in Eastern Africa and provides a target for manipulation to improve palatability.