Ocimum (basil) is a widespread genus commonly consumed for culinary and medicinal purposes attributed to unique secondary metabolites. The present research investigated the metabolic profiles of two Ocimum suspension cultures derived from O. basilicum and O. americanum , following elicitation by methyl jasmonate at different time intervals (24, 48, and 72 h) compared to the baseline at T = 0 h identifying bioactive metabolites and the influence on their expressional behaviour. Based on comprehensive extraction and mass spectrometry (MS)-based analytical protocols, the results showed that phenolic acid abundances, particularly rosmarinic acid, were increased significantly in O. americanum culture after 72 h to a 2.0-fold increase (p < 0.05). Similarly, its 3- O -methyl derivative was increased by 1.6-fold after 48 h in O. basilicum , and drastically (21.9-fold) after 72 h in O. americanum. Moreover, 23 out of the 56 identified bioactive secondary metabolites showed variable importance in the projection (VIP) scores ≥ 1, including isovitexin and 2,3-dihydroxybenzoic acid for O. basilicum. Furthermore, analysis of primary metabolites indicated that aromatic amino acids and sugars decreased indicating higher rates of consumption, while organic acids as end products of metabolisms increased significantly following elicitation for O. basilicum. Besides, the lipid profile showed a total of 107 metabolites. However, the effect of elicitation was variable between both species. Hence, the integration of untargeted metabolomics with chemical elicitation may improve our knowledge regarding the effects on various metabolites accumulation behaviour of different basil suspension cultures in a comparative and time-dependent approach. [Display omitted] • A metabolomic-based study for Ocimum sp. following chemical elicitation was performed. • O. basilicum showed different accumulation behaviours of most metabolites following elicitaion. • Around 56 bioactive metabolites, in addition to other metabolites were identified in Ocimum suspension cultures. • Production increase of phenolic metabolites is notably observed. • The study results may be further employed for optimization of certain metabolites production. [ABSTRACT FROM AUTHOR]