This study investigates the pharmacological potential of heterocyclic compounds, with a focus on quinolines, for combating infectious diseases. We explored the microwave-assisted synthesis of a series of pyran-based chloroquinoline derivatives and evaluated their antimicrobial properties through both in silico and in vitro methods. The successful synthesis of these derivatives was confirmed through spectral analysis. In silico analysis revealed that one of the synthesized compounds exhibited multiple hydrogen bonds with target proteins such as β-lactamase (PDB ID: 1AXB), PhoQ (PDB ID: 3CGZ), SsaV (PDB ID: 7AWA), FtsZ (PDB ID: 2VAM), and TasA (PDB ID: 5OF1), with the lowest binding energies recorded at -13.2, -12.2, - 15.1, -15.4, and -12.2 kcal/mol, respectively. Additional promising compounds demonstrated significant docking scores against selected protein targets. The in vitro antimicrobial efficacy was assessed using the well diffusion method and minimum inhibitory concentration (MIC) determination. Based on the well diffusion method, certain compounds exhibited larger zones of inhibition, while others showed moderate activity against S. enterica and B. subtilis. MIC analysis further identified the most effective compounds, with one showing an MIC value of 250 μg/ml, another <100 μg/mL, and another 500 μg/mL, while the remaining tested compounds displayed moderate effectiveness. This research highlights the potential of pyran-based chloroquinoline derivatives as promising therapeutic agents, demonstrating robust pharmacological properties and favorable antimicrobial profiles.