Matthiola longipetala subsp. livida is an annual herb in Brassicaceae that has received little attention despite the family’s high reputation for health benefits, particularly cancer prevention. In this study, UPLC-HRMS-MS analysis was used for mapping the chemical constituents of different plant parts (i.e., flowers, leaves, and roots). Also, spectral similarity networks via the Global Natural Products Social Molecular Networking (GNPS) were employed to visualize their chemical differences and similarities. Additionally, the cytotoxic activity on HCT-116, HeLa, and HepG2 cell lines was evaluated. Throughout the current analysis, 154 compounds were annotated, with the prevalence of phenolic acids, glucosinolates, flavonol glucosides, lipids, peptides, and others. Predictably, secondary metabolites (phenolic acids, flavonoids, and glucosinolates) were predominant in flowers and leaves, while the roots were characterized by primary metabolites (peptides and fatty acids). Four diacetyl derivatives tentatively assigned as O-acetyl O-malonyl glucoside of quercetin (103), kaempferol (108 and 112), and isorhamnetin (114) were detected for the first time in nature. The flowers and leaves extracts showed significant inhibition of HeLa cell line propagation with LC50 values of 18.1 ± 0.42 and 29.6 ± 0.35 µg/mL, respectively, whereas the flowers extract inhibited HCT-116 with LC50 24.8 ± 0.45 µg/mL, compared to those of Doxorubicin (26.1 ± 0.27 and 37.6 ± 0.21 µg/mL), respectively. In conclusion, the flowers of M. longipetala are responsible for the abundance of bioactive compounds with cytotoxic properties.
