Novel insights on the valorisation of onion (Allium cepa L.) are required to decipher its potential as nutritional and health-promoting agent. Wood vinegar (WV) was exogenously applied to onion plants to determine its effectiveness as an elicitor. An untargeted UHPLC-QTOF-MS metabolomics approach was combined with evaluating the bioactivities of onion extracts. WV promoted tissue and dose-specific effects on onion metabolome, involving both primary and secondary metabolites. Accordingly, the antioxidant and enzyme-inhibiting properties of onion were significantly enhanced after WV treatment. Moderate to high WV concentrations, ranging 250–500 µL L−1, were found optimal at conferring the highest rates of biological activities. Polyphenols were related to the bioactivities of onion extracts since phenolic acids, flavanols, and flavonols were suggested as the contributors to their health-enhancing properties. This approach provides insight into the characterisation of WV as a natural elicitor and opens wide perspectives on the research of onion tissues as biofactories of functional ingredients.