In an attempt to find a new class of antimicrobial agents, a series of fused chromenone derivatives containing pyridine and quinoline moieties were prepared via the reaction of 4-chloro-2-oxo-2H-chromene-3-carbaldehydes with cyclopentanone or cyclohexanone or a-tetralone in the presence of ammonium acetate in refluxing glacial acetic acid. The target molecules were also synthesized using interchange component strategy. These compounds were screened for their antibacterial activity against Gram-positive bacteria (Staphylococcus aureus and Streptococcus pyogenes), Gram-negative bacteria (Pseudomonas phaseolicola and Pseudomonas fluorescens), and antifungal activity against Fusarium oxysporum and Aspergillus niger. The compounds exhibited good to moderate antibacterial and antifungal properties against tested microorganisms. Structures of the newly synthesized compounds were established by elemental analysis and spectral data.