Abstract : Nigella sativa is an important medicinal plant found in several countries and used in folk medicine. Extract standardization is a crucial step in ensuring the quality and consistency of herbal extract used in medicinal therapy. The aim of this study was to determine the physicochemical content on the Nigella sativa extract and the antioxidant activity. DPPH methods were used to evaluate the antioxidant activity. Colorimetric methods were used to evaluate the secondary metabolite of the extract. Heavy metal contents, mineral and element contents were analysed by atomic absorption spectrophotometry. The thymoquinone content were analysed by HPLC. All the standardization methods used were based on the Indonesia Herbal Pharmacopeia and the General Standard Parameter of Extract. The 70% alcohol extract prepared with seed collected in Tawangmangu, Central Java, Indonesia. The yield of Nigella sativa extract was 1.34%, total acid-soluble ash content was 0.68%, Cd content 0 ppm, Pb content 0.09 ppm, and Cu content 0.02 ppm. The ethanolic extract of Nigella sativa contained alkaloids, saponins, tannins, and flavonoids related to the biological activities which are important to corroborate the different uses of this species. Interestingly, under HPLC analysis the content of thymoquinone was high around 6.96 mg/L of Nigella sativa extract. In addition, Nigella sativa extract has medium antioxidant activity with IC50 value of 908 ppm. The antioxidant activity is frequently associated with the high number of phenolic compounds found in these plants. In conclusion, the present physicochemical studies reveals that the Nigella sativa could be a potent source of antioxidant agent.