Antimicrobial resistance represents a major global public health challenge, with infections caused by multidrug-resistant pathogens such as MRSA being a core difficulty in clinical anti-infective therapy. Traditional fluoroquinolones are severely limited by cross-resistance and inadequate biofilm eradication. The 1,8-naphthyridin-4-one-3-carboxylic acid scaffold is privileged for quinolone antibacterials, and novel benzo[b]naphthyridinone-3-carboxylic acid derivatives offer a key strategy to overcome resistance. Herein, we optimized two complementary synthetic routes for the preparation of benzo[b]naphthyridinone-3-carboxylic acid derivatives, and systematically evaluated their antibacterial activity, safety profiles and mechanism of action using standard microbiological assays and molecular modeling. We synthesized eight 1,8-naphthyridinone derivatives, nine benzo[b]naphthyridinone derivatives and 85 key intermediates, with a shortest 6-step route overcoming the long-standing bottleneck of lengthy synthetic steps for this series. Lead compound 13kb displayed potent broad-spectrum activity (MIC = 0.08–0.63 μg/mL against Staphylococcus aureus, Escherichia coli, MRSA and Shigella flexneri), outperforming positive controls. It showed low cytotoxicity and hemolysis, concentration-dependent bactericidal effects, mature biofilm elimination and efficient DNA gyrase binding. This study provides efficient synthetic methodology and promising drug candidates for novel antibacterial development.