Display omitted •Six C-3′-C-6″ type of biflavonoids were isolated from S. trichoclada.•RF-A showed the strongest activity against MCF-7 cells among all isolated compounds.•RF-A induced the accumulation of ROS and Lipid peroxidation in MCF-7 cells.•RF-A induced ferroptosis in MCF-7 cells via the upregulation of VDAC2 protein. Breast cancer is one of the major malignant tumors in females, and currently, recurrence and metastasis are the main obstacles preventing effective breast cancer treatment. Biflavonoids of secondary metabolites from plants are excellent anticancer agents to fight sensitive and resistant breast cancer cell lines. In this study, six C-3′-C-6″ biflavonoids, including one new robustaflavone A (1, RF-A) and five known robustaflavone derivatives (2–6), were isolated from Selaginella trichoclada for the first time. We aimed to evaluate the inhibitory effects of compounds 1–6 against human breast cancer MCF-7 cells. Among the six compounds, RF-A showed the strongest activity, decreasing cell viability with an IC50 value of 11.89 μΜ. Furthermore, RF-A strikingly induced MCF-7 nonapoptotic cell death through ferroptosis by enhancing the expression of VDAC2 channels and reducing the expression of Nedd4 E3 ubiquitin ligase, leading to lipid peroxidation and ROS production. The results suggested that RF-A has potential as a novel breast cancer treatment through its regulation of the mitochondrial VDAC2 and Nedd4 pathways.