The cost-effective and environmental friendly biosynthesis of metal nanoparticles is becoming increasingly important in biomedical science. This study biosynthesized zinc oxide nanoparticles (ZnO NPs) from pumpkin seed extract and assessed its anticancer activity by using different molecular biology experiments in human breast cancer (MDA-MB-231) cell lines. The well-known analytical techniques, such as UV-visible spectrophotometry, energy-dispersive X-ray spectroscopy, field emission scanning electron microscopy, and transmission electron microscopy (TEM), were used to confirm the synthesis and characterize the morphological and structural features of biogenic ZnO NPs. The formation of spherical nanoparticles with an average size of 50–60 nm was recorded using scanning electron microscopy and TEM. We observed the dose-dependent increase in cytotoxicity of ZnO NPs in the MDA-MB-231 cell line with a 50% inhibitory concentration of 10 µg/mL. Moreover, the ZnO NPs also showed significant cell morphology changes, apoptosis induction, and reactive oxygen species production at the highest tested concentration. The cellular adhesion and migration assay indicated cell death and inhibition in the migration of breast cancer cells in response to ZnO NPs’ treatment. These results clearly demonstrated the significant anticancer potential of ZnO NPs against the studied breast cancer cell line. However, an extension of this study is recommended in different cancer models and, based on the results, in vivo validation should be done.