Direct-injection electron spray ionization mass spectrometry (DIESI-MS) can be used to quantify the whole set of positive and negative ions in complex biological samples. A cherry tomato cultivar was grown inside a greenhouse in soil pots supplemented with different nitrogen sources. Organic cultivation increased fruit dry matter while conventional chemical fertilizers increased yield due to higher water content. While soluble sugars were unaltered, secondary metabolism of tomato fruit was highly sensitive to compost soil supplied to the roots. From a total of ~1647 DIESI-MS signals, 725 revealed significant differences between treatments. Heatmap biclustering showed that ionomic differences were robustly maintained in independent experiments carried out during three consecutive years. The ionomic fingerprints allowed reproducible sample classification, reflecting the effect of organic farming on tomato fruit quality. Specific biomarker ions could be identified for various nitrogen sources. We propose DIESI-MS as an up-front strategy for plant food characterization aiming to identify the ions with the most significant differences across genotypes or agronomic conditions.