New benzo[b][1,4]thiazin-3(4H)-one derivatives (compounds 12a–p) 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]thiazin-3(4H)-ones showed, on the whole, potent toward all tested Gram-positive and Gram-negative microorganism (minimal inhibitory concentration ranging from 16 to 64 lg/ml), whereas weak effectiveness was exhibited against fungi. Data obtained suggested that 12g, 12i, and 12o exerted the best antibacterial activity against Grampositive bacteria and compound 12b demonstrated the best inhibition of Gram-negative bacteria. These observations provide some predictions to design further antimicrobial active compounds prior to their synthesis following with molecular modeling studies.