Spirodela polyrhiza (Araceae family) is a duckweed species that serves as a potential resource for feed, food, bioremediation, and pharmaceutical applications. In this study, we assessed the effects of different concentrations of melatonin (0, 0.1, 1, and 10 μM) on the growth of S. polyrhiza during in vitro culture and the metabolic profiles and productivities of useful metabolites using gas chromatography–mass spectrometry coupled with multivariable statistical analysis. We found that exogenous melatonin significantly improved the total dry weight and altered the metabolic profiles of S. polyrhiza cultures. Melatonin significantly enhanced the cellular production of useful metabolites, such as γ-aminobutyric acid, dopamine, threonine, valine, and phytosterols. The volumetric productivities (mg/L) of γ-aminobutyric acid, dopamine, campesterol, β-sitosterol, and stigmasterol were the highest in the presence of 10 μM melatonin on day 12. Moreover, the productivities of ascorbic acid and serotonin were the highest in the presence of 1 μM melatonin on day 12. Therefore, melatonin could be used to enhance the production of biomass and useful metabolites during large-scale S. polyrhiza cultivation in cosmetic, food/feed, and pharmaceutical industries.