Herein, a facile metal-free approach for the annellation of a pyran ring to thiobarbituric acid was described, and pyrano[2,3-d]pyrimidine 2 was obtained in a good yield. Subsequently, the valuable bicyclic pyranopyrimidine derivative 2 was utilized as a versatile building block for constructing assorted and hitherto unreported heterocyclic compounds. The reactivity of various carbon and nitrogen nucleophiles toward pyranopyrimidine derivative 2 was thoroughly investigated, leading to the synthesis of novel heterocycles with promising antimicrobial activity. Comprehensive characterization of the proposed structures of the newly synthesized fused pyranopyrimidine derivatives was carried out using IR, 1H NMR, 13C NMR, MS, and elemental analysis. Finally, the developed heterocycles were assessed in vitro for their antibacterial activity against selected bacterial strains. Additionally, the relationship between activity and structure was discussed. The results revealed very promising antibacterial properties for the majority of the synthesized heterocycles, indicating their potential as effective antibacterial agents.