This research presents a synthesis method for the CeO2/CuO nanostructured composite, which has potential applications as an antimicrobial material in the production of antimicrobial surface coatings, for example, for high-touch surfaces. The antimicrobial efficacy, mode of action, and potential cytotoxicity of CeO2/CuO towards the human immortalized keratinocyte cell line in vitro were studied compared to those of CuO, CeO2, and ionic Cu (a solubility control). The used synthesis method resulted in a CeO2/CuO nanostructured composite with a mean particle size of 27 nm and a specific surface area of 80.3 m2 g−1. The composite had a significant proportion (54%) of non-lattice oxygen species, highlighting the presence of substantial surface defects crucial for generating reactive oxygen species (ROS). The antimicrobial properties of CeO2/CuO, CuO, and CeO2 were assessed at six concentrations from 1 to 1000 mg L−1 in deionized water. The CeO2/CuO composite exhibited antibacterial efficacy at a minimum bactericidal concentration (MBC) of 100 mg L−1 towards Escherichia coli already after 2 h of contact and towards Pseudomonas aeruginosa and Staphylococcus aureus after 4 h of contact, whereas after 24 h of exposure, the antibacterial efficacy to all three bacterial strains was evident already at a MBC = 10 mg L−1. Fungi Candida albicans proved less susceptible than bacteria (24 h MBC = 100 mg L−1). Thus, the CeO2/CuO composite showed significant antibacterial efficacy against Gram-negative and Gram-positive bacteria, being at the same time safe to human keratinocytes in vitro in the case of which even 1000 mg L−1 caused no harmful effects after 2 h exposure and 500 mg L−1 caused no cytotoxicity after 24 h exposure. CeO2/CuO caused abiotic and biotic ROS production in all the tested environments. ROS production in deionized water was the most remarkable. Shedding of Cu-ions from CeO2/CuO was moderate and depended on the test environment, varying from 0.3 to 1 mg L−1, and considering the MBC of ionic Cu for microorganisms was not the main contributor to the antimicrobial activity of CeO2/CuO. The CeO2/CuO composite exhibited no acute toxicity to the environmentally relevant bacterium Vibrio fischeri. These findings indicate that CeO2/CuO's high ROS production is its primary antimicrobial mechanism and that due to its low cytotoxicity to human keratinocytes, it can be considered a promising antimicrobial agent.