The benzo[b][1,4]oxazin-3(4H)-one derivatives, 1a–p, carrying F, Br, and Cl on the benzene ring, or benzyl, cyclohexyl, n-hexyl, and tetrafuryl methylene groups attached to nitrogen atom were synthesized via Smiles rearrangement and assayed in vitro for their antimicrobial activity against Gram-positive, Gram-negative bacteria, and fungi. The antimicrobial activity of the benzo[b][1,4]oxazin-3(4H)-ones showed, on the whole, potency toward all the tested Gram-positive and Gramnegative microorganism (MIC ranging from 16 to 64 lg/ ml), whereas weak effectiveness was exhibited against fungi. Data obtained suggest that fluorine atom in the compounds, 1c, 1f, 1i plays an important role in enhancing the antimicrobial properties of this class of compounds. These observations provide some predictions to design further antimicrobial active compounds prior to their synthesis according to molecular modeling studies.