Eruca (Eruca sativa) seeds are valued as rich sources of macronutrients and phytochemicals, but their high erucic acid content may preclude their use in food applications, due to the health risks erucic acid. Therefore, this research aims to characterize the defatted Eruca seeds in terms of their chemical composition, phytochemical profile, antioxidant activity, functional properties, and the possibility of exploiting them in the production of functional biscuits with high phytochemical content and minimal erucic acid content. The results showed that defatted Eruca seeds flour (DESF) was extraordinarily rich in protein, carbohydrate, fiber, and ash contents with 38.78, 38.4, 9.13 and 5.33% (FW), respectively. The amino acids in Eruca protein were higher than those in the standard pattern except for threonine, methionine, and tryptophan. Erucic acid content in Eruca oil was 39.16 g/ 100 fat, and in DESF was 0.638 g/100 (FW). Whole Eruca seeds flour (WESF) extract showed greater antioxidant activity by about 1.24and1.53 times compared to DESF according to DPPH free radical-scavenging assay and FRAP assay, respectively. The most striking of phenolic compounds in both WESF and DESF is sinapic acid, which exceeds the amount of all other compounds that have been discovered. All studied functional properties were higher in DESF than in WESF. Finally, the results of the sensory evaluation indicate the possibility of replacing the DESF with up to 4% of wheat flour, with the improvement of most of the sensory acceptability parameters of the resulting biscuit, and the other sensory parameters not being negatively affected.