Due to the biological activities of pyrimidine and thienopyrimidine as antimicrobial agents, so in the present work, a series of new heterocyclic compounds containing thienopyrimidine moiety were synthesized such as, tetrazolo[1", 5": 1',6'] pyrimido[4',5': 4,5] thieno[2,3-d] pyrimidine derivatives (8-12b), pyrimido[3",2":1', 6'] pyrim-ido[ 4', 5': 4,5] thieno[2,3-d] pyrimidine derivatives (14,16) and pyrimido[5', 4': 4,5] thieno[2,3-e] pyrimido[1,2-c] [1,2,3] triazine derivative (15) by using 5-amino-4-phenyl-2-(p-tolylamino) thieno[ 2,3-d] pyrimidine-6-carbonitrile (7) as starting material. All synthesized compounds were evaluated for their antimicrobial activity. And Compounds (8-13) exhibited high antibacterial activity. Also, compounds (12b-18) exhibited high antifungal activity.