Abstract: A series of new bactericidal alginate calcium-copper nanoparticle nanocomposite films, denoted by Ca²⁺-Alg-Cu, were prepared by electrophoretic deposition with the constant current mode, by adjusting the copper nanoparticle composition in the electrolyte. According to the content of copper nanoparticles in the final formed film, they were denoted by Ca²⁺-Alg-Cu10, Ca²⁺-Alg-Cu20, and Ca²⁺-Alg-Cu50, respectively. The existence of copper nanoparticles in the nanocomposite films was confirmed via scanning electron microscopy (SEM), energy dispersive spectrometer (EPS), and Fourier-transformed infrared spectroscopy (FT-IR). Three different common infectious bacteria, including E. coli., S. aureus and P. aeruginosa, were taken as infectious pathogen models to explore the bactericidal property of Ca²⁺-Alg-Cu by plate counting method for 24 h. In addition, external animal skin cells such as mice L929 fibroblasts were incubated with Ca²⁺-Alg-Cu to assess the in vitro biocompatibility. Results show that Ca²⁺-Alg-Cu can potentially kill the three representative bacteria species by inducing cellular exterior membrane deformation and wrinkling, which is the inferred way of destruction in cellular structure. The potency of Ca²⁺-Alg-Cu depends on the mass fraction of copper nanoparticles (Cu) in the electrolyte. More efficiency can be obtained with higher content of copper nanoparticles, as can be seen in the experimental result. When the mass concentration of Cu in the electrolyte is lower than 0.4 mg/mL, the cellular viability is higher than 80%. Overall, it is indicated that a balanced antimicrobial activity and in vitro biocompatibility to the animal skin extracted cells can be realized.