Medicinal plants are rich sources of secondary metabolites. Lepidium sativum possess active compounds and secondary metabolites, including polyphenol, anthocyanin, flavonoid compounds, which are of special pharmaceutical and economic importance. Melatonin as bio-stimulator compound has a regulatory role on the amount of secondary metabolites and plant tolerance to environmental stresses. In this study, the effect of exogenous melatonin on secondary metabolites production on Lepidium sativum plants was carried out in a completely randomized design with 5 melatonin treatments (0, 5, 10, 50 and 100 μM) and 3 replications. After applying the treatment, the growth rate and content of the photosynthetic pigments, the leaf water content, as well as the content of flavonoids, anthocyanins and total phenolic compounds were investigated. Seed treatment with melatonin, especially at concentrations of 50 and 100 μM, improved the growth parameters and content of photosynthetic pigments. High levels of melatonin also increased the plant's secondary metabolites, including anthocyanins, carotenoids, and plant phenolic compounds. These effects can confirm the role of melatonin as a growth regulator and its impact on plant growth and resistance.