The increased prevalence of disease, mortality, and antibiotic resistance among aquatic microorganisms has renewed interest in non-conventional disease prevention and control approaches. Nanoparticles present several benefits in aquaculture and hold significant potential for controlling both human and animal infections. This study reports on the antibacterial properties of green copper oxide nanoparticles (CuO NPs) synthesized from the urine of Mithun (MU) (Bos frontalis). In addition, an array of analytical techniques, including scanning electron microscopy (SEM), X-ray diffraction (XRD), UV-visible spectroscopy (UV), and Fourier transform infrared spectroscopy (FTIR), were employed to investigate the synthesized MU-CuO nanoparticles.Aeromonas hydrophilaandAeromonas veronii, two bacterial fish pathogens known to cause severe infectious diseases in fish, were tested for their antibacterial efficacy against MU-CuO NPs. At 100 µg/mL, MU-CuO NPs exhibit enhanced antibacterial efficacy against two bacterial pathogens commonly found in fish. Applications in aquaculture may be looked at given that MU-CuO NPs showed greater antibacterial activity.