3-Cyano-5-ethoxycarbonyl-6-methyl-4-(2′-thienyl)-pyridine-2(1H)-thione (1) is synthesized and reacted with chloroacetamide or chloroacetonitrile to give 3-amino-5-ethoxycarbonyl-6-methyl-4(2′-thienyl)-thieno[2,3-b]pyridine-2-carboxamide 3a or its 2-carbonitrile analog 3b, respectively. Cyclocondensation of 3a with triethylorthoformate produced the corresponding pyridothienopyrimidineone 4, which on heating with phosphorus oxychloride gave 4-chloropyrimidine derivative 5. Compound 5 was used as key intermediate for synthesizing compounds 6, 9, 10, 11, and 12 upon treatment with some nucleophilic reagents such as thiourea, 5-phenyl-s-triazole-3(1H)-thione, piperidine, morpholine, or hydrazine hydrate, respectively. Reaction of pyridothienopyrimidinethione 6 with N-(4-tolyl)-2-chloroacetamide or ethyl bromoacetate afforded the corresponding S-substituted methylsulfanylpyrimidines 7 or 8. The condensation of 3b with triethylorthoformate gave azomethine derivative 13, which was reacted with hydrazine hydrate to give ethyl 3-amino-3,4-dihydro-4-imino-7-methyl-9-(2′-thienyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine-8-carboxylate (14). Compounds 12 and 14 were used as precursors for synthesizing other new thienylpyridothienopyrimidines as well as isomeric thienyl-s-triazolopyridothieno- pyrimidines. All synthesized compounds were characterized by elemental and spectral analyses such as IR, ¹H NMR, and ¹³C NMR. In addition, majority of synthesized compounds were tested for their antifungal activity against five strains of fungi. Moreover, compounds 3a, 5, 6, 8, and 22 were screened for their anticancer activity against HEPG-2 and MCF-7 cell lines.