A new series of 2-thiophenoxyquinoline-based penta-substituted pyridine derivatives, 6(a–r), has been synthesized by base-catalyzed cyclocondensation reaction through multi-component reaction (MCR) approach. In vitro antimicrobial activity of the synthesized compounds was investigated against a representative panel of pathogenic strains, specifically three Gram-positive bacteria (Streptococcus pneumoniae, Bacillus subtilis, Clostridium tetani), three Gram-negative bacteria (Escherichia coli, Salmonella typhi, Vibrio cholerae) and two fungi (Aspergllus fumigatus, Candida albicans). Majority of the compounds were found to be equipotent or more potent than that of the standard drugs.