Calcium oxide nanoparticles (CaO NPs) have unique catalytic and biological properties; their activities are highly influenced by their morphology; as a result, these characteristics are most needed for various applications in several fields, including material science, environmental science, and medicinal science. The primary motivation for synthesizing CaO NPs using a biological method is to suppress the usage of hazardous chemicals used in making its process, which will be more cost-effective and ecologically profitable. However, due to the complexity of the biological extracts employed in chemical processes, large-scale manufacturing of nanoparticles via the green synthesis approach remains a significant problem. As a result, the production of CaO NPs utilizing Moringa oleifera plant leaves aqueous extract as an alternative biological agent for capping, stabilizing, and reducing agents due to rich phytochemical parameters in synthesis was investigated in this study. The structural characterization of the CaO NPs obtained by using UV-Vis, FTIR, XRD, and SEM-EDS indicates the presence of purity and primarily aggregated spherical nanosized material with an average size of 32.08 nm observed. The XRD study revealed that heat annealing increased the size of the crystallites, favoring monocrystalline. Finally, these findings, together with the cheap cost of synthesizing the plant-mediated CaO NPs produced, show good antimicrobial (gram-positive) activities.