A series of 3-substituted amino-4,5-tetramethylene thieno[2,3-d] [1,2,3]-triazine-4(3H)-ones have been synthesized and characterized by UV,IR, 1H NMR, elemental and mass spectral analysis. The title compounds were evaluated for their antimicrobial activity by agar diffusion method against four bacteria and three fungi using Ampicillin and Miconazole nitrate as standards. The compounds VIIIa, IXa, Xa and XIa showed an antimicrobial efficacy considerably greater than the compounds Ia to VIIa with -H, phenyl and electron donating (activating) groups like methyl, ethyl and tolyl substitutions at R, suggesting that lipophillic groups like chloro, fluoro substitution on the phenyl ring plays an important role in enhancing the antimicrobial properties of this class of compounds. From the screening results it can be concluded that the compounds having the lipophillic groups like chlorophenyl and fluorophenyl groups at R exhibited appreciable antimicrobial activities. Whereas, the compounds are having -H, phenyl and electron donating (activating) groups like methyl, ethyl and tolyl substituents at R were less active against all the organisms used.