In this work, the combination of elicited plant suspension cultured cells (PSCCs) with untargeted metabolomics establishes a powerful, cutting-edge strategy to unravel the effects of elicitors on the biosynthetic potential of medicinal plants, as Bryophyllum sp. The PSCC technology constitutes a successful biotechnological system for the study and production of bioactive compounds throughout the inclusion of elicitors with the ability to modulate secondary metabolism. The use of methyl jasmonate (MJ) and salicylic acid (SA) as abiotic elicitors on bryophyllum PSCCs, resulted in differential effects on cell growth and secondary metabolism, depending on the species, including synergistic and antagonistic effects. This fact suggests that both elicitors play a pleiotropic effect on plant secondary metabolism, showing complex interactions, according to the UHPLC-QTOF mass spectrometry profiling. Thus, the combination of both elicitors induced a strong synergistic on B. daigremontianum PSCCs, with 2272 putatively annotated compounds, whereas it caused a negative effect on the secondary metabolism of B. × houghtonii PSCCs, being MJ the only elicitor driving a positive effect, presenting 2972 annotated compounds. Meanwhile, B. tubiflorum PSCCs did not show a significant modulation of secondary metabolism, with 1521 annotated compounds. The metabolite annotation indicated that three families of secondary metabolites were mainly affected by elicitation: phenolic compounds constituted the most affected family by elicitation, mainly represented by flavonoids and lignans; N-containing compounds included glucosinolates, amines, and alkaloids, reported to Bryophyllum sp. for the first time; and terpenoids included mainly phytoalexins and saponins. The results depict a deep genotype-dependent metabolomic reprogramming of secondary metabolism in response to elicitors, thanks to the application of untargeted metabolomics. This knowledge will allow the consideration of Bryophyllum sp. as a valuable source of bioactive compounds, with the potential associated to PSCCs, for being included in food, cosmetic, and pharmaceutical applications. © 2021 Elsevier B.V.