A novel series of benzotriazole derivatives were synthesized and characterized by NMR, IR and MS spectra. The bioactive assay manifested that most of the new compounds exhibited moderate to good antibacterial and antifungal activities against the tested strains in comparison to reference drugs chloromycin, norfloxacin and fluconazole. Especially, 2,4-dichlorophenyl substituted benzotriazole derivative 6f displayed good antibacterial activity against MRSA with MIC value of 4 𝜇g/mL, which was 2-fold more potent than Chloromycin, and it also displayed 3-fold stronger antifungal activity (MIC = 4 𝜇g/mL) than fluconazole (MIC = 16 𝜇g/mL) against Beer yeast. The preliminary interactive investigations of compound 6f with calf thymus DNA revealed that compound 6f could effectively intercalate into DNA to form compound 6f–DNA complex which might block DNA replication to exert antimicrobial activities. Molecular docking experiments suggested that compound 6f projected into base-pairs of DNA hexamer duplex forming two hydrogen bonds with guanine of DNA. The theoretical calculations were in accordance with the experimental results.