Background and Objective: This study aimed to determine the antibacterial activity of either zinc oxide or copper oxide nanoparticles alone or combined against Gram-positive and Gram-negative bacteria. Materials and Methods: In this experimental study Escherichia coli (E. coli ATCC 25922), Staphylococcus aureus (S.aureus ATCC 25923), Pesudomonas aueroginosa (P. aueroginosa ATCC 27853) and Staphylococcus Epidermidis (S. Epidermidis PTCC 1114) were used as test microorganisms. The Zinc and copper oxide nanoparticles were prepared commercially. The antibacterial activity of nanoparticles were studied using bacteriological tests such as minimum inhibitory concentration (MIC) and time kill study. Time kill studies were done using MIC nanoparticle concentration in mono and combined together mood for all the test microorganisms. The fractional inhibitory concentration (FIC) index used to define or to describe nanoparticle interactions at combined together mood of two nanoparticles. The ANOVA test (SPSS ver. 16) was used to compare the antibacterial activity of nanoparticles alone and combined. A P-value of ≤0.05 was considered significant. Results: The MIC value for E. coli and S.epidermidis against the combined nanoparticles had a lower concentration than mono metallic nanoparticles. By contrast, the MIC value for P.aeroginusa, and S. aureus against the combined mood of the nanoparticles had higher concentration. The fractional inhibitory concentration (FIC) index for E. coli, S.aureus, P. aueroginosa and S. epidermidis were 0.75, 9, 6, and 0.625, respectively. The results show that the Gram-negative bacteria seem to be more resistant to ZnO nanoparticles than the Gram-positive bacteria. Moreover, the antibacterial effect of nanoparticles in both mono and combined modes were time dependent (P<0.0001). Conclusion: The used commercial CuO/ZnO nanoparticles have great antibacterial potential against all of the strains, and the combination of zinc oxide and Copper oxide nanoparticles increases their bactericidal effect only for certain strains but not all.