Nigella sativa is one of the medicinal plant species that gained popularity for a wide range of medicinal applications due to its seeds which are rich in phytoconstituents. Continuous scientific investigations on N. sativa seeds are needed to better understand its many medicinal potentials. This will also form a composition-based foundation that support several old and/or new case beneficial histories of its seeds. In this study, the antimicrobial activity of N. sativa seeds was phytochemically characterized and evaluated. Different extracts of N. sativa seeds were obtained by maceration and soxhlet extraction methods using different extraction solvents. The obtained extracts were tested using UV-Vis, FTIR, TLC, and GC-MS techniques. Antimicrobial analysis against pathogenic bacterial strains (E. coli, P. aeruginosa, S. aureus and B. subtilis) was carried out by disc diffusion method using different preparations of N. sativa seeds. The screening analysis revealed the presence of all the tested phytochemicals. FT-IR analysis of N. sativa seeds oil extracted with absolute ethanol revealed functional groups that are associated with active ingredients of medicinal value. The GC-MS chromatograms revealed different chemical constituents whose known bioactivities and/or applications are essential in the management of life-threatening infections. Different extracts of N. sativa seeds showed antimicrobial activity with different efficacy against the tested pathogenic bacterial strains. Therefore, this study shows that extracts of N. sativa seeds contain a variety of chemical components and functional groups linked to their antimicrobial properties, and they might be natural precursors of nutraceuticals.