The excellent antimicrobial and anticancer efficacy of zinc oxide nanoparticles (ZnO NPs) with strong adsorption capability, low toxicity, low-cost and biocompatibility have spun these nanomaterials into one of the drug delivery systems. Herein, we prepared ZnO NPs with different morphologies and particle size using different methods. ZnO NPs were characterized by using Fourier transform infrared spectroscopy, X-ray diffraction, dynamic light scattering, zeta potential analyzer, scanning electron microscopy and transmission electron microscopy. Spheres with different average diameter ranged from 27 to 94.3 nm, flowers like shape with an average diameter of 250 nm, rod like shape with an average diameter of 92 nm, average length of 337 nm and a semispherical nanosheets with an average diameter of 49 nm were obtained. The nanoparticles were loaded by 5-fluorouracil as anticancer drug model. Adsorption capacity is influenced by the morphology and the size of nanoparticles. Kinetics using pseudo-first-order, pseudo-second-order and intra particle diffusion models was studied. Adsorption isotherms was explored using Langmuir, Freundlich and Temkin isotherm equations. Antibacterial activity of ZnO NPs onto gram-negative and gram-positive bacteria was studied by using disk diffusion agar method. The release of 5-fluorouracil from loaded ZnO NPs was studied at different pH media (2, 7.4) by using UV–visible spectrophotometer. 5-fluorouracil loaded ZnO NPs were examined against colon cancer (HCT) cell line. The spheres and flower-loaded drug showed higher efficacy than pure drug against colon cancer cell line. The results suggest that changing ZnO NPs morphology and particle size will affect antibacterial, anticancer activity, loading capacity of 5-fluorouracil and release profile.