A series of substituted 5,6,7,8-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyrimidine-4-one derivatives were newly synthesized starting from 5,6,7,8-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyrimidine-4-one derivatives (1). Furthermore, their derived tetrazolyl as well as the N-substituted derivatives were also prepared. The antimicrobial activity of the prepared compounds against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Aspergillus niger and Candida albicans were evaluated. The substituted thienopyrimidine derivatives 4 and 6 as well as the arylidine 10 were the highly active compounds.