A novel class of substituted pyridyl coumarin derivatives has been synthesized starting from 3-acetyl coumarin and chromone-3-carbaldehyde via one pot reaction and fully characterized by spectral and elemental analysis. All these derivatives 3a–l were screened in vitro for antimicrobial activity against a representative panel of pathogenic strains. Compounds demonstrated good to excellent antibacterial activity, while some compounds exhibited equipotent antifungal activity as compared to that of first line standard drug. As a part of investigation of new antitubercular agents, in vitro screening of synthesized compounds against Mycobacterium tuberculosis H37Rv has been done. Among the designed molecules, three compounds showed relatively better activity. From the entire study, it has been revealed that compounds appear to be better antimicrobials but relatively poor antituberculars.