ABSTRACT This work intended to study differences in oil yield and co-extraction of antioxidant and antibacterial compounds of Nigella sativa L. seed oil as a function of two main extraction processes: mechanical pressing and solvent extraction, including hexane and chloroform. Next, we studied such oils’ chemical composition and antioxidant and antibacterial activities. Notably, the chemical cartelization was conducted by use of HPLC. The antioxidant activity of the oils was assessed using DPPH, TAC, and FRAP assays. Antimicrobial activity was investigated against bacteria and fungi with clinical importanceT. HPLC-DAD analysis identified some bioactive compounds in the oils, including thymoquinone, carvacrol, and gallic acid. Results showed that oils extracted by different methods exhibit varying levels of antioxidant activity, with a higher concentration of antioxidant compounds in chloroform-extracted oils. Antimicrobial tests revealed significant inhibitory effects of the studied oils on the growth of bacteria and fungi. In conclusion, this research has made it possible to characterize the oils extracted from Nigella sativa L. Seeds by determining their chemical composition and biological properties, notably their antioxidant, antibacterial, and antifungal activity. These results contribute to a better understanding of the potential benefits of these oils for human health and open up exciting prospects for their use in various fields, notably as food additives and therapeutic agents.