Zinc oxide nanoparticles (ZnO NPs) were produced using the root hair extract of Phoenix dactylifera, and characterized by UV–Vis absorbance spectrophotometer, X-ray diffraction, particle size analyzer, and Fourier-transform infrared spectroscopy. Their antimicrobial activity and anticancer cytotoxicity were studied. An optimal nano-size range of 30.87–47.89 nm was obtained using 0.6 M of dihydrating zinc acetate salt and the root hair extract in a ratio of 1:2, respectively. ZnO NPs were observed to be around 45% more cytotoxic than doxorubicin (DOX) alone. Particularly, triple-negative breast cancer (TNBC) cells were observed to be more vulnerable to ZnO NPs than DOX alone which significantly reduced the viability of cancer cells to 9.01%. In addition, ZnO NPs were noticed to be 82.26% cytotoxic to lung cancer cells (A549). While testing ZnO powder did not cause any cytotoxicity on cancerous cells. Moreover, ZnO NPs showed promising antibacterial activity against different pathogenic organisms including Klebsiella pneumonia, Pseudomonas aeruginosa, Escherichia coli, Salmonella, and Staphylococcus aureus. Their activity was higher than the penicillin, gentamycin, and tetracycline based on the microbial inhibition zone. Generally, ZnO NPs demonstrate great potential for the chemotherapy of breast and lung cancer cells and bacterial infection.