Due to the resistance of some bacteria to antibiotics, research in the field of dealing with bacterial infections is necessary. A practical approach utilized in this study involves the preparation of an antibacterial thin film on the surfaces, which can effectively inhibit and reduce biofilm formation and bacterial adherence. In this study, we report the fabrication of bactericidal titanium (Ti) and copper (Cu) surfaces which involves a powerful co-sputtering method. This method provides a situation in which constituent elements are deposited simultaneously to control the composition of the thin film. Prepared samples were examined by energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), X-ray diffraction (XRD), atomic force microscopy (AFM), and contact angle measurements. To evaluate antibacterial behavior, we used two bacterial strains Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). Antibacterial activity of the prepared sample was assessed by determining the number of colony-forming units per milliliter (CFU/ml) using a standard viable cell count assay. Results indicated that as the Cu concentration increased, the nanoscale surfaces became rougher, with roughness values rising from 11.85 to 49.65 nm, and the contact angle increased from 40 to 80 degrees, indicating a hydrophilic character. These factors play a significant role in the antibacterial properties of the surface. The Ti-Cu films displayed superior antibacterial ability, with a 99.9% reduction (equivalent to a 5-log reduction) in bacterial viability after 2 h compared to Ti alone against both bacterial strains. Field emission scanning electron microscopy (FE-SEM) images verified that both E. coli and S. aureus cells were physically deformed and damaged the bacterial cell ultrastructure was observed. These findings highlight that adding Cu to Ti can improve the antibacterial ability of the surface while inhibiting bacterial adherence. Therefore, the Ti14-Cu86 sample with the highest percentage of Cu had the best bactericidal rate. Investigation of toxicity of Cu-Ti thin films was conducted the using the MTT assay, which revealed their biocompatibility and absence of cytotoxicity, further confirming their potential as promising biomaterials for various applications.