<![CDATA[The content of active compounds in natural materials is generally low and highly variable, thus requiring efficient and selective extraction methods. This study aims to evaluate a stepwise extraction process applied to the seeds, oil, and by-products of Nigella sativa (black cumin) to obtain thymoquinone (TQ) and other phytochemicals. The study consisted of four main stages: (1) extraction of HS oil using the cold press method, (2) stepwise extraction using 70% ethanol as solvent, (3) analysis of TQ content and phytochemical compounds in the extracts, and (4) evaluation of the antibacterial activity of habbatussauda oil extracts against pathogenic and non-pathogenic bacteria. Results showed that the cold press method yielded 34.46% oil. Stepwise extraction using 70% ethanol resulted in significant differences (p<0.05) in both yield and TQ content among the seed, oil, and by-product extracts. Among all samples, the ethanol extract from habbatussauda oil exhibited the highest yield and TQ concentration. Furthermore, at a concentration of 100 μg mL⁻¹, this extract inhibited the growth of pathogenic bacteria such as Salmonella typhimurium and Escherichia coli, without affecting non-pathogenic strains like Lactobacillus spp. and Bifidobacteria spp. In conclusion, stepwise extraction using 70% ethanol proved effective in enhancing TQ recovery and demonstrated potential as a phytobiotic agent with selective antibacterial activity against pathogenic bacteria. Key words: black seed, phytobiotic, phytochemicals, stepwise extraction, thymoquinone]]>
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