A series of azolylthioetherquinolones was synthesized and characterized by NMR, IR, MS and HRMSspectroscopy. All the newly prepared compounds were screened for their antimicrobial activities. Bioactive assay manifested that most of the azolylthioether quinolones exhibited good antimicrobial activities. Especially, imidazolylthioether quinolone 4e displayed remarkable anti-MRSA and anti-P. aeruginosa efficacies with low MIC values of 0.25 μg mL⁻¹, even superior to reference drugs. They induced bacterial resistance more slowly than clinical drugs. Molecular docking study indicated strong binding interactions of compound 4e with topoisomerase IV–DNA complex, which correlated with the inhibitory effect. The preliminarily interactive investigation of compound 4e with genomic DNA isolated from MRSA revealed that compound 4e could intercalate into MRSA DNA through a copper ion bridge to form a steady 4e–Cu²⁺–DNA ternary complex which might further block DNA replication to exert the powerful bioactivities.