Green nanoparticle synthesis is considered the most efficient and safe nanoparticle synthesis method, both economically and environmentally. The current research was focused on synthesizing zinc oxide nanoparticles (ZnONPs) from fruit and leaf extracts of Citrullus colocynthis. Four solvents (n-hexane, methanol, ethyl acetate, and aqueous) were used to prepare the extracts from both plant parts by maceration and extraction. Zinc acetate was used to synthesize the nanoparticles (NPs), and color change indicated the synthesis of ZnONPs. X-ray diffraction, UV spectroscopy, and scanning electron microscopy were used to study the ZnONPs. UV–visible spectroscopy revealed an absorbance peak in the 350–400 nm range. XRD patterns revealed the face-centered cubic structure of the ZnONPs. SEM confirmed a spherical morphology and a size range between 64 and 82 nm. Phytochemical assays confirmed that the complete flavonoid, phenolic, and alkaloid concentrations were higher in unrefined solvent extracts than in nanoparticles. Nanoparticles of C. colocynthis fruit aqueous extracts showed stronger antioxidant activity compared with the crude extracts. Strong antifungal activity was exhibited by the leaves, crude extracts, and nanoparticles of the n-hexane solvent. In a protein kinase inhibition assay, the maximum bald zone was revealed by nanoparticles of ethyl acetate extracts from leaves. The crude extracts and nanoparticles of leaves showed high cytotoxic activities of the n-hexane solvent, with LC50 values of 42.08 and 46.35, respectively. Potential antidiabetic activity was shown by the n-hexane (93.42%) and aqueous (82.54%) nanoparticles of the fruit. The bioactivity of the plant showed that it is a good candidate for therapeutic use. The biosynthesized ZnONPs showed promising antimicrobial, cytotoxic, antidiabetic, and antioxidant properties. Additionally, the in vivo assessment of a nano-directed drug delivery system for future therapeutic use can be conducted based on this study.