Most of the nearly 1500 species in Opuntia genus contain health-promoting components.These species are able to grow under adverse conditions, not suitable for other fruits/vegetables. Despite the acknowledge interest of their xoconostle fruits, especially due to their exquisite taste, other botanical parts from Opuntia are being scarcely studied. Herein, it was intended characterize Opuntia spp. seeds for their composition in soluble sugars, organic acids, tocopherols and phenolic compounds, besides evidencing their potential as sources of essential fatty acids. In addition, their antioxidant activity was assessed through different in vitro biochemical assays. Seeds of Opuntia spp. presented phytochemical profiles that allow their classification as a high-interest by-product. The levels of linoleic acid, γ-tocopherol and phenolic acids, as also their high antioxidant activity, are amongst the top characteristics that might endorse Opuntia spp. seeds in novel applications in food or pharmaceutical industries.