Nigella damascena L., a botanical species native to the Mediterranean region, is known for its biosynthesis of a spectrum of secondary metabolites including alkaloids, terpenoids, and flavonoids. These chemical constituents, present in both seed extracts and essential oils, exhibit a wide range of pharmacological properties including antimicrobial, molluscicidal, estrogenic, analgesic, antipyretic, and anti-inflammatory activity. In particular, the essential oil profile is predominantly characterized by the presence of β-elemene and damascenine, an alkaloid, which have been the subject of extensive research into their biological functionalities. Genetic engineering, particularly through genetic transformation, offers opportunities to gain new knowledge for a better understanding of biological processes and then to improve crop traits. The use of Agrobacterium-mediated (Rhizobium-mediated) transformation is a common method for the delivery of vectors with T-DNA constructs containing genes of interest or other elements required for genome editing, usually accompanied by selection or reporter genes such as uidA (gus) coding for β-glucuronidase. The refinement of transformation protocols has enabled the efficient delivery of complex DNA constructs. This chapter describes a systematic protocol for the successful genetic transformation of Nigella using Agrobacterium. These efforts resulted in a remarkable transformation efficiency of 67%, as determined by GUS staining of callus clumps. As a complementary step in this experimental framework, the successful regeneration of N. damascena plants is also demonstrated.