Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in folate biosynthesis, and a major malarial drug target. Point mutations in P. falciparum TS-DHFR have caused widespread global antifolate resistance, and yet the most effective antifolate known to overcome drug-resistance, WR99210, has poor oral bioavailability. More specific, less toxic therapies are urgently needed. Antifolates target the conversion of methylene tetrahydrofolate to dihydrofolate by TS, and that of dihydrofolate to tetrahydrofolate by DHFR. In humans, TS and DHFR are two discrete enzymes. In P. falciparum, however, TS-DHFR is a bifunctional enzyme, with TS and DHFR encoded within a single protein, and tethered together with by a ‘linker’ region. This linker is not homologous to any other known TS or DHFR enzymes, and is essential for enzyme activity. This bifunctional enzyme thus presents different design approaches for developing novel inhibitors against drug-resistant mutants: developing active-site inhibitors equally effective against wildtype and drug-resistant parasites, or targeting unique non-active site regions for parasite-specific inhibitors. As a first step in identifying unique inhibitors, we performed a high-throughput in silico screen of a database of diverse, drug-like molecules against a non-active site pocket within the linker region of TS-DHFR. The top compounds from the virtual screen were evaluated by enzymatic and cellular assays. In vitro enzymatic studies and cell culture studies of wildtype and drug-resistant P. falciparum parasites identified three compounds active to 20 μM IC50s in both wildtype and antifolate-resistant enzymatic studies, as well as in P. falciparum cell culture. Moreover no inhibition of human DHFR enzyme was observed indicating the inhibitory effects appeared to be parasite-specific. Notably, all three compounds had a biguanide scaffold. Further computational analysis was utilized to determine the relative free energy of binding and these calculations suggested that the compounds might preferentially interact with the active site over the screened ‘linker’ region. To resolve the two possible modes of binding, co-crystallization studies of the compounds complexed with TS-DHFR enzyme were performed to determine the three-dimensional structures. Surprisingly, the structural analysis revealed that these novel, biguanide compounds, distinct from WR99210, do indeed bind at the active site of DHFR, and additionally revealed the molecular basis by which they overcome drug-resistance. To our knowledge, these are the first co-crystal structures of novel, biguanide, non-WR99210 compounds that are active against folate-resistant malaria parasites in cell culture. These studies reveal how serendipity coupled with computational and structural analysis can identify unique compounds as a promising starting point for rational drug design to combat drug-resistant malaria.