The current study report a convenient, simple, and low cost approach for the biogenic synthesis of CuO/Fe3O4 nanocomposites (NCs) from pumpkin seeds extract and their vitro cytotoxicity. The characterization of finally obtained CuO/Fe3O4 nanocomposites (NCs) performed using UV-Visible, FT-IR, XRD, XPS, GC-MS, SEM-EDX and TEM analysis. The formation and elemental analysis were determined using the energy-dispersive X-ray (EDX) microanalysis technique. The formation of rod-like monoclinic and spherical, having size range 5 nm-20 nm confirmed by scanning electron microscope (SEM) and transmission electron microscopy (TEM) respectively. Finally, the MTT assay of the synthesized composites was evaluated for toxicity against cancerous cell lines HCT-116 (Colon cancer cell) and A549 (human lung adenocarcinoma cell). The synthesized composite material showed moderate (IC50 = 199 μg/mL) to low (IC50 = 445 μg/mL) activity against HCT-116 and A549 cell lines, respectively.