Pyrimidoquinolinone-derived compounds have exhibited a broad spectrum of biological activities and have been acknowledged as a potential antimalarial scaffold through multiple conventional strategies. In the present study, a novel series of 2,4-dimethoxy-5,8,9,10-tetrahydropyrimido[4,5-b]quinolinones 4(a-m) were synthesized via a one-pot three-component reaction of cyclohexane-1,3‑dione, 4-amino-2,6-dimethoxypyrimidine and various aldehyde with l-proline as the catalyst and acetic acid as the solvent under reflux condition. In vitro antimalarial activity toward P. falciparum 3D7 strain cultures were investigated. Compounds 4b and 4m demonstrated significant efficacy against strains of P. falciparum, with IC50 values of 0.63 and 0.65 μg/mL, respectively. The results were also supported by molecular docking and ADME assays.