Nigella sativa, an annual herbaceous flowering plant of the Ranunculaceae family, is considered an important medicinal plant due to the presence of several bioactive compounds in its seeds, including both volatile and non-volatile compounds. The cultivation of numerous genotypes of N. sativa are witnessed in different parts of the world with varying compositions of such chemical compounds. Since the variation in composition determines the quality grade of the seeds, this study was carried out to explore the compositional variation of twelve different genotypes of N. sativa cultivated in Central Queensland, Australia. The results showed total phenolic content (TPC), FRAP and CUPRAC (antioxidants), and thymoquinone in the range of 291–529 mg GAE/100 g DW, 703–966 mg TE/100 g DW, 2533–3416 mg TE/100 g DW, and 219–349 mg/100 g DW, respectively. The highest value of TPC, thymoquinone, FRAP and CUPRAC was observed in genotype AVTKS#E, AVTKS#F, AVTKS#4 and AVTKS#D, respectively. The lowest value of TPC and FRAP was observed in genotype AVTKS#24 and the CUPRAC and thymoquinone was lowest in genotype AVTKS#23 and AVTKS#1, respectively. Monomeric anthocyanins were absent in the methanolic seed extracts of all nigella genotypes. There was a strong positive correlation among the TPC, CUPRAC and FRAP. However, despite thymoquinone being reported as a strong antioxidant in the literature, there was no significant correlation of thymoquinone with TPC or CUPRAC, and only a weak positive correlation with FRAP. Overall, the genotypes with comparatively higher value of thymoquinone, TPC and antioxidant capacity (both, FRAP and CUPRAC) showed particular potential for breeding programs.