Background: 1,3-benzoxazine is a group of compounds with various applications in medicine, including sedative, cardiovascular, neuropsychiatric treatments, and antiviral properties. Additionally, these compounds effectively combat many types of bacteria and fungi. In this study, we focus on the derivatives of 6-iodophenyl-3H-benzo[e][1,3]oxazine-2,4-dione and evaluate their antibacterial activity. Objectives: Synthesize the derivatives of 6-iodophenyl-3H-benzo[e][1,3]oxazine-2,4-dione and assess their antibacterial activity. Materials and methods: The subjects of this study are the derivatives of 6-iodophenyl-3H-benzo[e][1,3]oxazine-2,4-dione, synthesized from the starting material 5-iodosalicylic acid through two steps: forming the amide bond and synthesizing 6-iodophenyl-3H-benzo[e][1,3]oxazine-2,4-dione. The structures of the derivatives were determined using melting point, UV spectrum, IR spectrum, MS spectrum, and ¹H-NMR, ¹³C-NMR spectrum. The antibacterial activity was tested using the agar diffusion method and dilution method. Results: The 6-iodophenyl-3H-benzo[e][1,3]oxazine-2,4-dione derivatives were successfully synthesized with an average yield of  73.91%. All three derivatives exhibited antibacterial activity against the tested bacterial strains. Conclusion: The three derivatives of 6-iodophenyl-3H-benzo[e][1,3]oxazine-2,4-dione are relatively strong antibacterial compounds (MIC < 2 µg/ml) and have potential clinical applications.