In the present study, some new 2-(pyridin-2-yl)thieno[2,3-d]pyrimidin-4(3H)-ones derivatives (IIa-o) were synthesized. The target compounds (IIa-o) were synthesized through the acid catalyzed condensation of 2-cyano, 3-cyano, and 4-cyano-pyridines with various 2-amino-3-carbethoxythiophenes (Ia-e). All thiophene derivatives were synthesized by Gewald reaction. The structures of the newly synthesized compounds were confirmed by UV-Visible, FT-IR, (1)H-NMR, and mass spectral studies. All synthesized compounds were evaluated for their antimicrobial activity against various gram-positive and gram-negative bacterial and fungal strains. Amongst the synthesized compounds IIa, IIb, IId, IIe, and IIm were found to be active.