Cyclization 4-hydrazinothieno[2,3-d]pyrimidines with 1,1'-carbonyldiimidazole was proposed as the novel and highly effective approach for synthesis of the series of thieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidin-3(2H)-ones. Heterocyclic systems obtained were modified by alkylation with chloroacetamides (DMF-K2CO3), and produced 2- (3-oxothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidin-2(3H)-yl)-N-acetamides. Antimicrobial activity screening for all of the compounds obtained has been performed by the agar well diffusion method. The highest antimicrobial activity was determined for ethyl 9-methyl-3-oxo-2,3-dihydrothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine-8-carboxylate 2d, which showed the activity against Proteus vulgaris similar to Synthomycine. Some of the compounds also showed better activity than Metronidazole against the strain of Candida albicans fungi.