Roselle (Hibiscus sabdariffa) is a plant valued for its leaves, seeds, and especially its flowers (calyx and corolla), which are important for their nutritional and medicinal properties. The red petals are commonly used in West Africa to make jams, gels, ice creams, and soft drinks. However, overharvesting of the flowers threatens the plant’s reproduction and existence. To address this, the biotechnological production of natural phytochemicals through callus culture can optimize the production of these valuable compounds without seasonal constraints. This study focused on enhancing the pharmacological value of Roselle by identifying phytochemical compounds in its petals and evaluating their biological activities. Roselle petal extract contains 18 phenolic compounds, including phenolic acids (chlorogenic, protocatechuic) and flavonoids (gossypetrin, sabdaretin, gossypetin, luteolin, gossytrin, hibiscetin, rutin, myricetin, eugenol, nicotiflorin, quercitrin, quercetin, kaempferol, astragalin, cynaroside), with sabdaretin, gossypetin, hibiscetin, and quercetin being the predominant flavonoids. Anthocyanins such as delphinidin 3-O-sambubioside and cyanidin 3-O-sambubioside are also notable. The Roselle callus culture produced 23 phytochemical compounds, including ten flavonoids, seven phenolic acids, and six anthocyanins, with certain phenolic acids and anthocyanins synthesized exclusively in the callus. The diverse physiological properties of these phenolic compounds allow for a range of biological activities such as anti-allergic, anti-inflammatory, antimicrobial, and cardioprotective effects.