Present study reports the development of novel pyridopyrimidine derivatives as biotin carboxylase inhibitors with potent antibacterial activity. These compounds were designed to avoid possibility of resistance development. Accordingly eighteen compounds were synthesised and characterized on the basis of spectral data. These compounds were tested for their antibacterial potential by the enzyme kinetic assay against the biotin carboxylase. The minimum inhibitory concentration (MIC) and single step resistance studies were also performed. Compound 2-((2-Phenylpyrido[2,3-d]pyrimidin-4-yl)amino)phenol (6o) showed promising activity in biotin carboxylase inhibition with low MIC. It showed molecular docking score of -7.96, this compound showed formation of hydrogen bonds with the active site residues and van Der Walls intractions. The MIC of compounds under investigation was in the rage of 2-5µg/mL over most of the strains studied. It also showed the mutant selection windows of around five which is better than the reference compound rifampin. This compound 6o can be studied further and developed into a potential antibacterial lead molecule.