Manihot esculenta Crantz (cassava) of Euphorbiaceae family is one of the well-known crops that possesses a broad range of therapeutic characteristics due to the rich content of vitamins, minerals and active secondary metabolites. The present work was planned to scrutinize the probable effect of different elicitor concentrations on the stimulation of callus growth and metabolite production such as total carbohydrates, total phenol and total flavonoids along with the antioxidant activity assessment. In vitro, calli cultures were formerly produced from stem segments of cassava using MS medium containing 5 mg/l 2,4dichlorophenoxyacetic acid (2, 4-D) and 0.2 mg/l benzylaminopurine (BAP). Salicylic acid was more efficient for callus production compared to methyl jasmonate and glutathione. Calli cultures of cassava recorded the maximum carbohydrate content with glutathione at concentrations of 4 and 2 mM (38.69 and 35.06 mg glu g-1dw), respectively. Similarly, the highest total phenol content (28.74 and 28.35 mg GAE g-1dw) was obtained using glutathione at concentrations of 4 and 2 mM, respectively. Whereas the highest content of flavonoids (1.73 mgQEg-1dw), was achieved with 2 mM glutathione. The lowest carbohydrate, phenols and flavonoid contents were detected in the control calli cultures (11.62 mg glu g-1dw; 0.44 mgGAEg-1dw and 0.16 mgQEg-1dw respectively). The highest free radical scavenging activity of calli cultures against DPPH free radicals has resulted in MeJ20 of 96.73%, while the lowest was in the control (free elicitor) medium of 80.21%. Although varied concentrations of elicitor were used in the present experiments, it can be concluded that the calli treated with elicitors had a higher metabolite content compared to the control. The obtained results indicated that with the appropriate type and concentration of elicitor, an improved metabolite content of Manihot esculenta Crantz can be achieved and hypothesized as a rich source of biological components to be useful in nutritional supplements and pharmaceuticals.