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 is seen 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 values of TPC, thymoquinone, FRAP, and CUPRAC were observed in genotypes AVTKS#E, AVTKS#F, AVTKS#4, and AVTKS#D, respectively. The lowest values of TPC and FRAP were observed in genotype AVTKS#24, and the CUPRAC and thymoquinone were 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 values of thymoquinone, TPC, and antioxidant capacity (both FRAP and CUPRAC) showed particular potential for breeding programs.