Abstract: Melatonin has been reported in a variety of food plants and, consequently, in a number of plant-derived foodstuffs. In grapevine (Vitis vinifera L.) products, it was found in berry exocarp (skin) of different cultivars and monovarietal wines. Herein, we assessed, by means of mass spectrometry, the occurrence of melatonin in all berry tissues (skin, flesh, and seed) at two different phenological stages, pre-véraison and véraison. We detected the highest melatonin content in skin, at pre-véraison, whereas, at véraison, the highest levels were reported in the seed. Furthermore, during ripening, melatonin decreased in skin, while increasing in both seed and flesh. The relative concentrations of melatonin in diverse berry tissues were somewhat different from those of total polyphenols (TP), the latter measured by the Folin-Ciocalteau assay, and more abundant in seed at pre-véraison and in exocarp at véraison. The highest antiradical activity, determined by both DPPH (2,2-diphenyl-1-pycryl hydrazyl) and ABTS [(2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] radical-scavenging assay, was reported at pre-veráison in seed. To the best of our knowledge, we reported, for the first time, the occurrence of melatonin in grape seeds.