Nigella sativa L. (black cumin) is a widely used medicinal and nutritional crop, yet its chemical characteristics under Albanian agroecological conditions remain unexplored. This study provides the first assessment of the fatty acid profile and macronutrient composition of N. sativa L. varieties cultivated in Tirana, with the aim of supporting breeding efforts, functional food development, and crop diversification. Eight varieties originating from different countries were grown at the Experimental Didactic Field of the Agricultural University of Tirana. Mature seeds were analyzed for fatty acids following derivatization to FAMEs using GCMS-QP2010 Ultra, while macronutrient concentrations (Na, K, Ca, Mg, P) were quantified using ICP-MS. The varieties exhibited significant inter-varietal variability. T7 showed the highest monounsaturated fatty acids (21.9%) and calcium concentration (2,012.31 mg kg⁻¹). T8 recorded the highest saturated fatty acids (17.2%) and sodium level (128.58 mg kg⁻¹), whereas T4 had the highest polyunsaturated fatty acids (63.0%). For minerals, T3 contained the greatest potassium (11,450.19 mg kg⁻¹) and magnesium (2,904.45 mg kg⁻¹), while T5 had the highest phosphorus content (9,309.01 mg kg⁻¹). These results highlight substantial nutritional diversity within N. sativa L. grown under Tirana conditions, identifying promising genotypes with superior fatty acid and mineral profiles. Such genotypes have strong potential for use in functional foods, nutraceutical production, and sustainable agricultural development.