A series of novel 2-(2-oxo-2H-chromen-3-yl)-5H-chromeno[4,3-b]pyridin-5-ones 4a–4l have been synthesized using two methodologies (using Mannich base of 4-hydroxy coumarin and 4-chloro-3-formyl coumarin) (Patel et al., 2012) and assayed for their antibacterial activity against gram-positive bacteria viz. Bacillus subtilis (MTCC 441), Staphylococcus aureus(MTCC 96) and gram-negative bacteria viz. Escherichia coli (MTCC 443), Salmonella typhimurium (MTCC 98). The compounds 4a–4l were also assayed for their antifungal activity against Aspergillus niger (MTCC 282) and Candida albicans (MTCC 227). All the compounds 4a–4l exhibited potent inhibitory activity against gram-positive bacteria compared to standard drugs at the tested concentrations. The compounds also showed appreciable activity against gram-negative bacteria as well as fungi. The compounds 4e, 4f, 4i, 4k, and 4l are found to be the most proficient members of the series and emerged as potential molecules for further development.