© 2020, Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.The aim of this study was to determine the effects of 24-epibrassinolide (24-eBL) and l-phenylalanine (l-phy) on the root growth, total phenolics, total flavonoids, and caffeic acid derivatives’ (CADs) accumulation in Agrobacterium rhizogenes-mediated hairy roots of Echinacea purpurea. For this aim, different concentrations of 24-eBL (0.5, 1.0 and 2.0 mg L−1) and l-phy (100, 500, and 1000 µM) were applied to hairy roots and the roots were harvested five times at 10 day intervals. After harvest, hairy roots were evaluated for fresh root weight, dry root weight, growth index, and the contents of total phenolics, total flavonoids, and CADs. The highest fresh root weight, dry root weight, and growth index were obtained from the roots treated with 0.5 mg L−1 24-eBL and harvested at 50 days after treatment, while l-phy had no significant influence on root growth. Among the 24-eBL applications, 1.0 mg L−1 24-eBL was found as the optimum concentration, providing the highest total phenolics, total flavonoids, cichoric acid, caftaric acid, echinacoside, and p-coumaric acid contents. On the other hand, 500 μM l-phy was determined as the most suitable l-phy treatment for giving the maximum values in terms of all investigated parameters. In terms of harvesting period, 50th day was the most appropriate harvest time for secondary metabolite accumulation. Consequently, the results showed that 24-eBL and l-phy treatments can be successfully increased the production of secondary metabolites in transgenic hairy roots of E. purpurea and 24-eBL implementations could be a more effective strategy when compared to l-phy.