A new series of 5-amino-6-(benzo[d]thiazol-2-yl)-2-(2-(substitutedbenzylidene)hydrazinyl)-7-(4-chlorophenyl)pyrido[2,3-d]pyrimidin-4(3H)-one derivatives (7a–k) were synthesized. All the newly synthesized compounds were screened for their in vitro antibacterial activity, against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus pyogenes and for antifungal activity against Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Penicillium marneffei and Mucor. Compounds 7b, 7e, 7f, 7g, 7h and 7j showed excellent in vitro antibacterial activity and antifungal activity than the standard drugs. All the compounds were characterized by IR, 1H NMR, 13C NMR, LCMS mass and C, H, N analyses.