Infectious diseases caused by drug-resistant bacteria represent one of the most significant global public challenges of this century. There is an urgent need for the treatment of drug-resistant Gram-negative bacterial infections. A series of 3,4-dihydro-2H-[1,3]oxazino[5,6-h]quinoline derivatives were synthesized and evaluated for their antibacterial activity against Gram-negative bacteria including strains from ATCC and clinical isolates, initially revealing the structure-activity relationship. Among them, 22 compounds demonstrated inhibitory activity (MICs: 3.125-12.5 μg/mL) against Escherichia coli (E. coli) ATCC 25922 and Acinetobacter baumannii (A. baumannii) ATCC 19606. Among these, 7 compounds exhibited good inhibitory activity against MDR A. baumannii clinical isolates, with MICs ranging from 3.125 to 12.5 μg/mL. Most of these compounds also showed lower cytotoxicity than IMB-881. Notably, 2 compounds, 4n1 and 4b3, significantly extended the survival of Galleria mellonella larvae infected with E. coli. Mechanism studies have revealed that compounds 4n1 and 4b3 might disrupt the interaction between LptA and LptC, showing moderate affinity for LptA protein. These compounds also induce abnormal bacterial morphology and cause outer membrane damage. This finding provides a novel class of antibiotic sensitizers with the potential to effectively fight against E. coli and A. baumannii.