The study was carried out to investigate the interactive effects of exogenous melatonin and excess amounts of zinc and copper on the growth and physiological parameters, antioxidant defense system and nutritional balance of cannabis seedlings. Cannabis sativa L. plants, grown under a completely randomized design, were irrigated with complete Hoagland’s nutrient solution. CuSO ₄ (0, 50 and 150 µM) and ZnSO ₄ (0, 50 and 100 µM) and their combinations were supplied to 21-day-old seedlings for 2 weeks. During the second week, melatonin was added to the nutrient solution at 100 μM. Zn and Cu stress led to reduced growth and physiological parameters, it promoted oxidative stress, changes in antioxidant enzymes activity and imbalance of mineral nutrients in cannabis seedlings. However, melatonin alleviated the growth retardation and physiological disorders of seedlings under normal conditions and heavy metal stress. The content of reduced glutathione and the activity of antioxidant enzymes such as glutathione reductase and ascorbate peroxidase were improved by melatonin. Excess amounts of zinc and copper changed the pattern of nutritional elements distribution in cannabis seedlings. Cu and Zn caused reduced content of Fe, Ca and K ions in shoots and roots. Melatonin treatment was able to adjust the nutrients content in metal-stressed seedlings up to the level of the control. Exogenous melatonin reduced toxic levels of Cu and Zn in seedlings overloaded with copper and zinc. MT also raised K, Ca and Fe concentrations in roots and shoots of seedlings under stress. Our results support the idea that melatonin acts as a powerful antioxidant, it can also be considered as a potent regulator of ion homeostasis in cannabis seedlings under heavy metal toxicity. Further studies still need to investigate the alleviatory effects of melatonin under field conditions.