A series of novel quinazolinone azoles were synthesized and characterized by NMR, IR, MS and HRMS spectra. Bioactive assay showed that some target compounds exhibited significant antimicrobial potency. Especially, nitroimidazole derivative 3a displayed comparable or even better antibacterial efficacies (MIC = 0.03–0.05 µmol/mL) in contrast with norfloxacin (MIC = 0.01–0.05 µmol/mL) and chloromycin (MIC = 0.02–0.10 µmol/mL). The preliminary interactive investigations of compound 3a with calf thymus DNA by UV-vis spectroscopic method revealed that compound 3a could bind to DNA to form compound 3a-DNA complex by an intercalative mode and further block DNA replication to exert their powerful antibacterial and antifungal activities. The hydrogen bonds and van der Waals forces played important roles in the association of compound 3a-HSA.