A series of isoflavone functionalized 3,4-dihydro-1,3-oxazine hybrids was synthesized in good to excellent yields through a Mannich-type condensation cyclization reaction of 6-chloro-7-hydroxy-3-(2-methoxy-phenyl)-chromen-4-one or 6-chloro-7-hydroxy-3-(2-methoxy-phenyl)-chromene-4-thione with formaldehyde and primary amines. After spectroscopic characterization, these newly prepared hybrids were evaluated for their antibacterial activities against two of each Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains. Among the screened compounds, dihydro[1,3]oxazine-fused 4- thionoisoflavones(9band9c) exhibited potent inhibitory activity against all the tested bacterial species. Moreover, compound 9b possessed most promising antibacterial activity against P. aeruginosa and B. subtiliswith MIC 16 μg/mL and S. aureusand E.coliwith MIC 32 μg/mL. Further, 9bdemonstrated better efficacy (MIC=16 μg/mL) than the standard drug ampicillin (MIC= 32 μg/mL) against P. aeruginosaand it also found to be equipotent (MIC=16 μg/mL) as ampicillin against B. subtilis. Considering the disk diffusion and synergistic studies, 9be�merged asmost active compound showing potent activity against all the tested bacterial strains. In addition, no significant hemolysis or cytotoxicity was observed towards human embryonic kidney (HEK293)cells as well as Galleria mellonellalarvae (in vivo). Hence, compound 9bhas potential to be further explored alone and in combination with ampicillin as a next generation antibacterial agent.