Malaria remains a significant global health concern causing numerous fatalities and the emergence of antimalarial drug resistance highlights the urgent need for novel therapeutic options with innovative mechanisms of action and targets. This study aimed to design potential inhibitors of 6-pyruvoyltetrahydropterin synthase (PTPS), synthesize them, and experimentally validate their efficacy as antimalarial agents. A structure-based approach was employed to design a series of novel derivatives, including amidinyl, amidoximyl and hydroxamic acid analogs (, , , and ), with a focus on their ability to bind to the Zn present in the active site of PTPS. The syntheses of these compounds were accomplished through various multi-step synthetic pathways and their structural identities were confirmed using H and C NMR spectra, mass spectra, and elemental analysis. The compounds were screened for their antiplasmodial activity against the NF54 strain of and cytotoxicity testing was performed using L-6 cells. The acute toxicity of the compounds was evaluated in mice. Docking studies of the compounds with the 3D structure of PTPS revealed their strong binding affinities, with compound exhibiting notable metal-acceptor interaction with the Zn in the protein binding pocket thereby positioning it as a lead compound for PTPS inhibition. The antiplasmodial studies revealed moderate efficacies against the NF54 strain, particularly compounds and which displayed IC < 0.2 μM. No significant cytotoxicity was noted on the L-6 rat cell line. Moreover, studies suggested that compound exhibited both safety and efficacy in treating rodent malaria. The identified lead compound in this study represents a possible candidate for antimalarial drug development and can be further explored in the search for alternative antifolate drugs to combat the malaria menace.