Antimicrobial resistance (AMR) has emerged as a significant and pressing public health concern, posing serious challenges to effectively preventing and treating persistent diseases. Despite various efforts made in recent years to address this problem, the global trends of AMR continue to escalate without any indication of decline. As AMR is well-known for antibiotics, developing new materials such as metal containing compounds with different mechanisms of action is crucial to effectively address this challenge. Copper, silver, and chitosan in various forms have demonstrated significant biological activities and hold promise for applications in medicine and biotechnology. Exploring the biological properties of these nanoparticles is essential for innovative therapeutic approaches in treating bacterial and fungal infections, cancer, and other diseases. To this end, the present study aimed to synthesize silver@copper oxide (Ag@CuO) nanoparticles and its chitosan nanocomposite (Chi-Ag@CuO) to investigate their antimicrobial efficacy. Various established spectroscopic and microscopic methods were employed for characterization purposes, encompassing scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Subsequently, the antimicrobial activity of the nanoparticles was assessed through MIC (minimum inhibitory concentration), MBC (minimum bactericidal concentration), and well-disk diffusion assays against , , , and . The size of the CuO-NPs, Ag@CuO, and Chi-Ag@CuO NPs was found to be 70-120 nm with a spherical shape and an almost uniform distribution. The nanocomposites were found to possess a minimum inhibitory concentration (MIC) of 5 μg/mL and a minimum bactericidal concentration (MBC) of 250 μg/mL. Moreover, these nanocomposites generated varying clear inhibition zones, with diameters ranging from a minimum of 9 ± 0.5 mm to a maximum of 25 ± 0.5 mm. Consequently, it is evident that the amalgamation of copper-silver-chitosan nanoparticles has exhibited noteworthy antimicrobial properties in the controlled laboratory environment, surpassing the performance of other types of nanoparticles.