The usage of active antimicrobial food packaging systems has become inevitable with the globalization of food trade, and in this regards antimicrobial nanocomposites have attracted universal attention. The present study aims to examine the antimicrobial effects of CuO-containing nanocomposite on two important spoilage bacteria, namely gram-positive Bacillus subtilis and gram-negative Enterobacter aerogenes, and comparison of its antibacterial effect with ZnO-containing nanocomposite. To synthesize the nanoparticles of CuO, sonochemical method has been employed. The nanoparticles have been characterized by X-ray diffraction. By melt mixing in a twin-screw extruder, nanocomposite film containing 2 wt% CuO nanoparticles was prepared. CuO-containing nanocomposites had reduced the growth of both bacteria. CuO-containing nanocomposite had a stronger antibacterial effect on both of the microorganisms in comparison with ZnO-containing nanocomposite, which could be ascribed to their small size. Due to the significant antibacterial effect of ZnO- and CuO-containing nanocomposites, they have the potential to be used in active food packaging.