Antimicrobial resistance is one of the biggest threats to public health across the globe. Bacteria, fungi, viruses, and protozoans have been exhibiting resistance against antimicrobial drugs making them ineffective. Hence, the development of new antibiotics with a different mode of action is highly desirable. In this study, 10 new chromone‐incorporated fused thiazolo[2,3‐b]quinazolinone derivatives, 8a‐j, have been prepared via Biginelli reaction involving aromatic aldehydes, 1‐tetralone, and thiourea followed by a reaction with 2‐chloro‐N‐phenylacetamide, and Knoevenagel condensation with 3‐formylchromone. All the structures of the compounds were characterized by NMR, FTIR, and mass spectrometry. The in vitro antibacterial activities of all the synthesized compounds against the four different microbial strains were evaluated. Among them, few compounds demonstrated prominent activity against Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. No appreciable activity of any compound against Klebsiella pneumoniae was observed. Molecular docking studies were employed to reveal the interactions responsible for the potent compounds' activities against S. aureus, S. pyogenes, and P. aeruginosa. Both in vitro and in silico studies have been carried out by using standard agar well diffusion protocol and Auto Dock Vina in PyRx. The results indicated that compound 8c was the potential compound as it showed good affinity toward the receptors of all three organisms. Molecular dynamics simulation of the 8c‐1JIJ complex for 100 ns further confirmed the potentiality of 8c. The pharmacokinetic properties of the compounds indicate that the studied molecules have exhibited a favorable profile.