In recent years, there is a growing interest towards the green synthesis of metal nanoparticles, particularly from plants; however, yet no published study on the synthesis of ZnO.NPs using the Deverra tortuosa extract. Through this study, zinc oxide nanoparticles (ZnO.NPs) have been synthesized based on using the environmentally benign extract of the aerial parts of D. tortuosa as a reducing and capping agent. ZnO.NPs synthesis was confirmed using UV-Visible (UV-Vis) spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD) and High Resolution-Transmission Electron Microscope (HR-TEM). The qualitative and quantitative analyses of plant extract were done. The potential anticancer activity was in vitro investigated against two cancer cell lines (human colon adenocarcinoma “Caco-2” and human lung adenocarcinoma “A549”) compared to their activities on the human lung fibroblast cell line (WI38) using the MTT assay. Both the aqueous extract and ZnO.NPs showed a remarkable selective cytotoxicity against the two examined cancer cell lines.