<p>In this study, Ca₃Co₄O₉ nanostructures were effectively produced using a novel one-step oil bath method. The produced nanoparticles’ morphological, structural, and antibacterial characteristics were systematically studied. The Ca₃Co₄O₉ phase with an average crystallite size of 27.4&#xa0;nm was established by X-ray diffraction (XRD). The functional groups associated to the Ca₃Co₄O₉ structure were acknowledged using Fourier transform infrared (FTIR) spectroscopy. Scanning electron microscopy (SEM) study of the surface morphology exposed that the nanostructures have a uniform spherical shape and an average particle size of about ~ 0.7&#xa0;μm. Spherical agglomerates are the effect of Ostwald ripening and aggregation-driven assembly, which is determined by the decrease of surface free energy. The antibacterial activity of the produced Ca₃Co₄O₉ nanostructures was estimated against two hazardous bacterial strains: Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. For S. aureus, the zone of inhibition measured 8.26 ± 0.25&#xa0;mm, while for E. coli, it was 8.99 ± 0.05&#xa0;mm. The spherical morphology’s high surface-to-volume ratio and many grain boundaries boosted its contact with bacterial cell membranes, promoting the generation of reactive oxygen species (ROS) and subsequent bacterial cell death. The findings indicate that Ca₃Co₄O₉ nanostructures are intriguing possibilities for biological applications, particularly in the development of pharmaceutical medications and antimicrobial medical devices, offering a sustainable means of combating microbial diseases.</p>