A series of novel naphthalimide-derived metronidazoles as new type of antimicrobial agents were for the first time designed, synthesized and characterized by NMR, IR and HRMS spectra. Experimental results revealed that most of them displayed moderate to good antibacterial activity towards Gram-positive and negative bacteria. Especially, compound 7b was able to not only exhibit effective inhibition towards the growth of P. vulgaris(MIC = 0.002 mmol/m L) and S. dysenteriae(MIC = 0.01 mmol/m L), but also have rapidly killing effect and prevent the development of bacterial resistance. Further research revealed that the highly active molecule 7b could not only intercalate into calf thymus DNA to form a steady supramolecular complex and thus might block DNA replication to exert the powerful bioactivities, but also be effectively transported by human serum albumin(HSA) via the formation of the 1:1supramolecular complex, in which hydrogen bonds and hydrophobic effect played important roles in the association of compound 7b with HSA. Molecular docking indicated that the supramolecular interactions between 7b and topoisomerase II were driven by hydrogen bonds. A series of novel naphthalimide-derived metronidazoles as new type of antimicrobial agents were for the first time designed, synthesized and characterized by NMR, IR and HRMS spectra. Experimental results revealed that most of them displayed moderate to good antibacterial activity towards Gram-positive and Negative bacteria. Especially, compound 7b was able to not only exhibit effective inhibition towards the growth of P. vulgaris (MIC = 0.002 mmol / m L) and S. dysenteriae (MIC = 0.01 mmol / m L), but also has accelerating kill effect and prevent the development of bacterial resistance. Further research revealed that the highly active molecule 7b could not only intercalate into calf thymus DNA to form a steady supramolecular complex and thus might block DNA replication to exert the powerful bioactivities, but also be strong human serum albumin (HSA) via the formation of the 1: 1 supramolecular complex, in which hydrogen bonds and hydrophobic effect plays important roles in the association of compound 7b with HSA. Molecular docking indicated that the supramolecular interactions between 7b and topoisomerase II were driven by hydrogen bonds.