ABSTRACT: Nigella species are widely used to cure various ailments. Their health benefits, particularly from the seed oils, could be attributed to the presence of a variety of bioactive components. Roasting is a critical process that has historically been used to facilitate oil extraction and enhance flavor; it may also alter the chemical composition and biological properties of the Nigella seed. The aim of this study was to investigate the effect of the roasting process on the composition of the bioactive components and the biological activities of Nigella arvensis and Nigella sativa seed extracts. Our preliminary study showed that seeds roasted at 50 °C exhibited potent antimicrobial activities; therefore, this temperature was selected for roasting Nigella seeds. For extraction, raw and roasted seed samples were macerated in methanol. The antimicrobial activities against Streptococcus agalactiae, Streptococcus epidermidis, Streptococcus pyogenes, Candida albicans, Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, and Klebsiella oxytoca were determined by measuring the diameter of the zone of inhibition. The cell viability of extracts was tested in a colon carcinoma cell line, HCT-116, by using a microculture tetrazolium technique (MTT) assay. Amino acids were extracted and quantified using an automatic amino acid analyzer. Then, gas chromatography-mass spectrometry (GC-MS) analysis was performed to identify the chemical constituents and fatty acids. As a result, the extracts of raw and roasted seeds in both Nigella species showed strong inhibition against Klebsiella oxytoca, and the raw seed extract of N.arvensis demonstrated moderate inhibition against S. pyogenes. The findings of the MTT assay indicated that all the extracts significantly decreased cancer cell viability. Moreover, N. sativa species possessed higher contents of the measured amino acids, except tyrosine, cystine, and methionine. The GC-MS analysis of extracts showed the presence of 22 and 13 compounds in raw and roasted N. arvensis, respectively, and 9 and 11 compounds in raw and roasted N. sativa, respectively. However, heat treatment decreased the detectable components to 13 compounds in roasted N. arvensis and increased them in roasted N. sativa. These findings indicate that N. arvensis and N. sativa could be potential sources of anticancer and antimicrobials, where the bioactive compounds play a pivotal role as functional components.