The rapid emergence of antimalarial drug resistance underscores the urgent need for new chemotypes targeting validated parasite enzymes. This study reports the design of six novel vanillin-based imidazole Mannich bases (1a-2c) as potential antimalarial agents targeting Plasmodium falciparum DHFR-TS through regioselective Mannich functionalization of an imidazole-vanillin scaffold. The target compounds were synthesized via a two-step sequence involving a Debus-Radziszewski condensation followed by regioselective Mannich amination, affording the desired products in high yields (60-95%) and excellent purity (94-99%). In vitro antiplasmodial evaluation against the P. falciparum 3D7 strain demonstrated potent inhibitory activity, with compound 1c exhibiting the highest potency (IC50 = 0.002 ± 0.00004 μM), surpassing chloroquine (IC50 = 0.022 ± 0.0006 μM). To elucidate the molecular basis of their biological activity, the synthesized compounds were further evaluated using DFT calculations, molecular docking, molecular dynamics simulations, MM/PBSA binding free energy analysis, and ADMET predictions. Molecular docking against wild-type PfDHFR-TS (PDB ID: 1J3I) revealed that compounds 1a-2c exhibited stronger binding affinities than compounds 2a-2c, WR99210, and chloroquine through favorable interactions within the catalytic pocket. Furthermore, 100 ns molecular dynamics simulations and MM/PBSA analysis confirmed the structural stability and favorable binding free energy of the PfDHFR-TS-1c complex. These findings identify vanillin-based imidazole Mannich bases as promising scaffolds for the development of new PfDHFR-TS-targeted antimalarial agents.