Selenium is an essential micronutrient and its insufficient intake has been recorded in Croatia and other parts of the world. Plants absorb selenium from the soil and are responsible for its primary accumulation in the food chain. For this reason, biofortification of plants with selenium is a useful way to ensure optimal intake of selenium in the population. Selenium nanoparticles are relatively novel form of selenium with hypothesized improved bioavailability and reduced toxicity compared to inorganic and organic forms of selenium. Biofortification with selenium can lead to optimal selenium content in plants for human consumption and increased antioxidant content, since it can affect the biosynthesis of phenolic compounds and other secondary metabolites in plants. In this paper, the antioxidant activity of arugula and spinach extracts biofortified with different chemical forms of selenium was investigated. The highest anti-radical and reductive potential was shown for arugula and spinach extracts biofortified with selenium nanoparticles functionalized with humic acid. All extracts were shown to be biocompatible with HepG2 cells. The protective antioxidant effect of the extract against pro-oxidants has also been proven. It was dependent on the chemical form of selenium used for biofortification. In the case of arugula, the greatest effect was shown for the extract of plants biofortified with selenium nanoparticles functionalized with polysorbate, while in spinach the greatest effect was seen for the extract of plants biofortified with selenium nanoparticles functionalized with humic acid. Biofortification had an impact on the antioxidative activity of the extracts and it was dependent on the applied chemical form of Se. Nanoparticles can be considered as a potential alternative to selenate in biofortification, since they significantly affected the antioxidant properties of arugula and spinach, which can consequently have a beneficial effect on human health.