A series of hitherto unreported pyrido-pyrimidine-2-ones/pyrimidine-2-thiones were synthesized under microwave assisted solvent free reaction conditions in excellent yields and evaluated in vitro for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes inhibitory activity. Among the pyridopyrimidine derivatives, 7e and 7l displayed 2.5- and 1.5-fold higher enzyme inhibitory activities against AChE as compared to standard drug, galanthamine, with IC50 of 0.80 and 1.37 μM, respectively. Interestingly, all the compounds except 6k, 7j and 7k displayed higher inhibitory potential against BChE enzyme in comparison to standard with IC50 ranging from 1.18 to 18.90 μM. Molecular modeling simulations of 7e and 7l was performed using three-dimensional structure of Torpedo californica AChE (TcAChE) and human butyrylcholinesterase (hBChE) enzymes to disclose binding interaction and orientation of these molecule into the active site gorge of respective receptors.