Echinacea (Echinacea purpurea L. Moench) is one of the most important medicinal plants worldwide with various pharmacological properties. In the current study, a biotic elicitation strategy using the fungal elicitors yeast extract (YE), Aspergillus niger and Fusarium oxysporum was employed to improve the productivity of callus biomass, total phenolics and total flavonoids in E. purpurea leaf-derived callus. Significant differences were recorded between biotic elicitors concerning callus production, total phenolics and flavonoids accumulation, DPPH scavenging activity, and antioxidant enzymes activity. Callus biomass, growth index, and relative growth rate were significantly enhanced in elicited cultures. The highest content of total phenolics was recorded with 4 g/l YE, 1 g/l A. niger or 0.25 g/l F. oxysporum. However, 0.25 g/l F. oxysporum was superior in enhancing the productivity of total phenolics, and the content and productivity of total flavonoids, representing 310.60%, 361.40% and 649.50% increase of control, respectively. In general, callus extracts exhibited 79.02 – 89.51% scavenging activity of DPPH comparing with ascorbic acid (97.91%). YE at 4 g/l recorded the highest activity of catalase (CAT) and peroxidase (POD) enzymes. However, adding A. niger or F. oxysporum have a superior effect on polyphenol oxidase (PPO) activity. The current study revealed the possibility of using callus cultures and biotic elicitation as a promising tool for the in vitro production of phenols and flavonoids phytoconstituents with high antioxidant capacity from the high-value medicinal plant E. purpurea.