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The present study reports the synthesis of ZnO-NPs using Acantholimon serotinum extracts followed by characterization and evaluation of biological activities. Field emission scanning electron microscope revealed irregular spherical morphology with a size in the range of 20–80 nm. The X-ray diffraction analysis confirmed the synthesis of highly pure ZnO NPs with a hexagonal shape and a crystalline size of 16.3 nm. The UV-Vis spectroscopy indicates the synthesis of ZnO-NPs. FT-IR confirmed the presence of phytocomponents in the plant extract, which was responsible for nanoparticle synthesis. According to MTT results, the biosynthesized ZnO-NPs showed cytotoxic effects on human colon cancer Caco-2 (IC₅₀: 61 µg/mL), neuroblastoma SH-SY5Y (IC₅₀: 42 µg/mL), breast cancer MDA-MB-231 (IC₅₀: 24 µg/mL), and embryonic kidney HEK-293 (IC₅₀: 60 µg/mL) cell lines. Significant reactive oxygen …