Abstract: The identification of melatonin in plants has inspired new investigations to understand its biological function and which endogenous and external factors control its levels in these organisms. Owing to the therapeutical and nutraceutical properties of melatonin, it should be important to develop reliable analytical methods for its quantification in vegetal matrices containing this indoleamine, such as grape and wine. The main objectives of the present study were to test whether melatonin levels fluctuate during the day in berry skins of Vitis vinifera L. cv Malbec, thereby possibly relating its abundance to its putative antioxidant function, to determine whether daylight reaching clusters negatively controls melatonin levels, and to evaluate whether total polyphenols and anthocyanins also change through a 24-hr period. Grapes were harvested throughout the day/night to determine the moment when high levels of these components are present in grapes. The presence of melatonin in grapes was evaluated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. It is shown for the first time that melatonin levels fluctuate during the day/night cycle in plants grown under field conditions in a fruit organ of the species Vitis vinifera. We also determined that the diurnal decay of melatonin in berry skins is induced by sunlight, because covered bunches retained higher melatonin levels than exposed ones, thus explaining at least part of the basis of its daily fluctuation. Evidence of melatonin’s antioxidant role in grapes is also suggested by monitoring malondialdehyde levels during the day.