The possibility of fabricating metal nanoparticles using plant extracts has already been explored in phytomedicines. Nanobiotechnology has a wide range of potential applications, including the cosmetics industry, packaging, coatings, biomedicine, and the enhancement of biological activity. Nickel oxide (NiO) was fabricated in a waste-free, low-cost method and then used to adorn Marsdenia tenacissima extract (MTE), a crucial plant leaf. After forming the nanoparticles, MTE@NiO-NPs were also tested for their optical and biological qualities. The morphological characteristics of NiO-NPs are rhombohedra and slightly agglomerated, and the particle size of the biosynthesized NiO-NPs is observed in the range of ∼ 15 nm. The in vitro cytotoxicity of Marsdenia tenacissima extract (MTE) and nanoparticles on lung cancer cells (A549 and H1299) by MTT assay. The in vitro cytotoxicity assay results revealed that the newly fabricated nanoparticles effectively kill cancer cells with low minimal inhibitory concentration. The various biochemical staining assays assessed the cell death and morphological investigation. Furthermore, the mode of cell death was evaluated by mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS). Therefore, the study concludes that the plant leaves extract of Marsdenia tenacissima is a powerful reducing/capping agent in fabricating MTE@NiO-NPs, demonstrating useful biological features.