Both 2-(N-arylcarbamoyl)methylsulfanyl-3-cyano-5-ethoxycarbonyl-6-methyl-4-(2′-thienyl)pyridines 2a-e and their isomers, 3-amino-2-(N-arylcarbamoyl)-5-ethoxy-carbonyl-6-methyl-4-(2′-thienyl)thieno[2,3-b]pyridines 3a-e were synthesized by reaction of ethyl 3-cyano-1,2-dihydro-6-methyl-4-(2′-thienyl)-2-thioxopyridine-5-carboxylate (1) with the respective N-aryl-2-chloroacetamides in the presence of different basic catalysts. Compounds 3a-e were used as precursors for synthesizing of pyridothienopyrimidinones 4a-e, pyridothienotriazinones 5a-e and tetrahydropyridothienopyrimidinones 6a-c via treatment with triethyl orthoformate, nitrous acid, and/or 4-chlorobenzaldehyde respectively. The photophysical properties of aminothienylthienopyridines 3a-e and tetrahydropyridothieno-pyrimidine-4(3H)-ones 6a-c were studied and the fluorescence data revealed that all selected compounds possess AIE behaviors in solution and in solid state. Most of the prepared compounds were evaluated in vitro for their antibacterial and antifungal activities, and considerable results were obtained. Moreover, the cytotoxic activity of thienopyridines 3a-d against MCF-7 and HepG2 cell lines was evaluated and they showed moderate to very strong activity.