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Modern nanotechnology encompasses every field of life. Nowadays, phytochemically fabricated nanoparticles are being widely studied for their bioactivities and biosafety. The present research studied the synthesis, characterization, stability, biocompatibility, and in vitro bioactivities of calcium oxide nanoparticles (CaONPs). The CaONPs were synthesized using Citrullus colocynthis ethanolic fruit extracts. Greenly synthesized nanoparticles had an average size of 35.93 ± 2.54 nm and showed an absorbance peak at 325 nm. An absorbance peak in this range depicts the coating of phenolic acids, flavones, flavonols, and flavonoids on the surface of CaONPs. The XRD pattern showed sharp peaks that illustrated the preferred cubic crystalline nature of triturate. A great hindrance to the use of nanoparticles in the field of medicine is their extremely reactive nature. The FTIR analysis of the CaONPs showed a coating of phytochemicals on their surface, due to which they showed great stability. The vibrations present at 3639 cm−1 for alcohols or phenols, 2860 cm−1 for alkanes, 2487 cm−1 for alkynes, 1625 cm−1 for amines, and 1434 cm−1 for carboxylic acids and aldehydes show adsorption of phytochemicals on the surface of CaONPs. The CaONPs were highly stable over time; however, their stability was slightly disturbed by varying salinity and pH. The dialysis membrane in vitro release analysis revealed consistent nanoparticle release over a 10-h period. The bioactivities of CaONPs, C. colocynthis fruit extracts, and their synergistic solution were assessed. Synergistic solutions of both CaONPs and C. colocynthis fruit extracts showed great bioactivity and biosafety. The synergistic solution reduced cell viability by only 14.68% and caused only 16% hemolysis. The synergistic solution inhibited Micrococcus luteus slightly more effectively than streptomycin, with an activity index of 1.02. It also caused an 83.87% reduction in free radicals.