Plasmodium falciparum thymidylate kinase (PfTMPK) is a key enzyme in pyrimidine nucleotide biosynthesis. 3-Trifluoromethyl-4-chloro-phenyl-urea- α-thymidine has been reported as an inhibitor of Mycobacterium tuberculosis TMPK (MtTMPK). Starting from this point, we designed, synthesized and evaluated a number of thymidine analogues as antimalarials. Both 5′-urea-α- and β-thymidine derivatives were moderate inhibitors of PfTMPK and furthermore showed moderate inhibition of parasite growth. The structure of several enzyme-inhibitor complexes provides a basis for improved inhibitor design. However, we found that certain 5′-urea-α-thymidine analogues had antimalarial activity where inhibition of PfTMPK is not the major mode of action. Optimization of this series resulted in a compound with potent antimalarial activity (EC = 28 nM; CC = 29 μM).