Copper and chitosan are used for biomedical applications due to their antimicrobial properties. In this study, a facile method for the synthesis of chitosan-copper oxide nanocomposites (nCuO-CSs) was modified, yielding stable colloidal nCuO-CSs suspensions. Using this method, nCuO-CSs with different copper-to-chitosan (50–190 kDa) weight ratios (1:0.3, 1:1, 1:3) were synthesized, their physicochemical properties characterized, and antibacterial efficacy assessed against Gram-negative Escherichia coli and Pseudomonas aeruginosa, and Gram-positive Staphylococcus aureus. The nCuO-CSs with a primary size of ∼10 nm and a ζ-potential of >+40 mV proved efficient antibacterials, acting at concentrations around 1 mg Cu/L. Notably, against Gram-negative bacteria, this inhibitory effect was already evident after a 1-h exposure and surpassed that of copper ions, implying to a synergistic effect of chitosan and nano-CuO. Indeed, using flow cytometry and confocal laser scanning microscopy, we showed that chitosan promoted interaction between the nCuO-CSs and bacterial cells, facilitating the shedding of copper ions in the close vicinity of the cell surface. The synergy between copper and chitosan makes these nanomaterials promising for biomedical applications (e.g., wound dressings).