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The objectives of this research were to carry out GC–MS and LC–MS-based phytochemical profiling of Barleria hochstetteri, as well as flow cytometry-based mechanistic investigations of the cytotoxic effect of its extracts against breast and lung cancer cell lines. This preclinical in vitro study was carried out in Saudi Arabia and India, from 11 August to 15 January 2022. Barleria hochstetteri was sequentially extracted using the Soxhlet extraction technique. Utilizing LC–MS and GC–MS methods, the phytochemical profiling was performed. Additionally, the total phenolic compounds and flavonoids were quantified in the plant extract using spectrophotometric techniques. In this study, we first examined the cytotoxicity of the plant extract on non-malignant L929 cells and on the carcinogenic MCF-7 and A549 cell lines. Then, we studied the underlying molecular pathways by means of Anti-Bcl-2, caspase-3, and DNA fragmentation (TUNEL) assays, using flow cytometry. The results revealed phenolic compounds and flavonoids to be the two major components in the methanolic extract of B. hochstetteri, with concentrations of 3210 µg GAE/g dwt and 1863 µg QE/g dwt, respectively. Results from GC–MS and LC–MS analyses revealed the presence of bioactive phytochemicals with known cytotoxicity. From the MTT assay on cell viability, the IC50 of the methanol extract for the MCF-7 and A549 cell lines were 219.67 and 144.30 µg/mL, respectively. With IC50 values of 324.24 and 266.66 µg/mL, respectively, the aqueous and methanol extracts were less toxic when tested against the non-cancerous L929 cell line. The extract caused early and late apoptosis in the tested breast and lung cancer cells by activating caspase-3 and inhibiting Bcl-2 protein, and it also caused cell death via DNA damage, based on flow cytometric and molecular marker analyses. These findings indicate that the methanol extract of B. hochstetteri was cytotoxic on breast cancer and lung cancer cell lines. To uncover cancer-fighting chemicals, there is a need for further research on B. hochstetteri, as it is a promising source of anti-cancer chemotherapeutic drugs. [ABSTRACT FROM AUTHOR]