A class of quinazolone thiazoles was identified as new structural scaffolds for potential antibacterial conquerors to tackle dreadful resistance. Some prepared compounds exhibited favorable bacteriostatic efficiencies on tested bacteria, and the most representative <b>5j</b> featuring the 4-trifluoromethylphenyl group possessed superior performances against <i>Escherichia coli</i> and <i>Pseudomonas aeruginosa</i> to norfloxacin. Further studies revealed that <b>5j</b> with inappreciable hemolysis could hinder the formation of bacterial biofilms and trigger reactive oxygen species generation, which could take responsibility for emerging low resistance. Subsequent paralleled exploration discovered that <b>5j</b> not only disintegrated outer and inner membranes to induce leakage of cytoplasmic contents but also broke the metabolism by suppressing dehydrogenase. Meanwhile, derivative <b>5j</b> could intercalate into DNA to exert powerful antibacterial properties. Moreover, compound <b>5j</b> gave synergistic effects against some Gram-negative bacteria in combination with norfloxacin. These findings indicated that this novel structural type of quinazolone thiazoles showed therapeutic foreground in struggling with Gram-negative bacterial infections.