Microbial infections and rising antimicrobial resistance highlight the need for new treatments. Benzoxazine derivatives are promising candidates due to their diverse biological activities. This study synthesized and evaluated the antimicrobial properties of 2H-benzo[b][1,4] oxazin-3(4H)-one derivatives. The synthesis involved reacting 2-aminophenol with chloroacetic acid, followed by sulfonation with chlorosulfonic acid and subsequent nucleophilic substitution with aryl amines. The compounds were tested against E. coli, S. aureus, and B. subtilis, with compound 4e showing the highest activity across all strains. Molecular docking studies targeting E. coli DNA gyrase revealed that compound 4d had the strongest binding affinity, followed by compounds 4a, 4e, and 4f. Binding interactions with key amino acid residues were analyzed, providing insights for further optimization. Compound 4e’s broad-spectrum activity and high potency suggest its potential as a new antimicrobial agent, warranting further in vivo testing and toxicity evaluation.