A series of new potentially multi-targeting antimicrobial 2-aminothiazolyl quinolones were designed, synthesized and characterized by 1H NMR, 13C NMR, IR, MS and HRMS spectra. Bioactive assay manifested that some of the prepared compounds showed moderate to good antibacterial and antifungal activities. Noticeably, compound 10f could effectively inhibit the growth of B. typhi and MRSA with MIC values of 1 and 8 μg/mL, respectively. Experimental results revealed that compound 10f was membrane-active and had the ability to rapidly kill the tested strains and effectively prevent the development of bacterial resistance. Moreover, this compound also exhibited low toxicity against L929 cells. Molecular docking indicated that compound 10f could bind with topoisomerase IV-DNA complexes through hydrogen bonds and hydrophobic interactions. Quantum chemical studies were also performed on 10f to understand the structural features essential for activity. The preliminary mechanism research suggested that compound 10f could intercalate into calf thymus DNA to form a steady supramolecular complex which might block DNA replication to exert the powerful bioactivities.