An NMR-based screen of a commercially available fragment library was performed on the folate pathway antimicrobial target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Staphylococcus aureus (SaHPPK). Initial 1D saturation transfer difference-NMR screening resulted in an impractically high hit rate (43 ), which advocated the use of a strategy based on 2D (SOFAST) N-15 HMQC NMR experiments. Chemical shift perturbations were used to identify, validate, and map the location of 16 initial binders (hit rate of 2 ). Fourteen compounds were purchased based on an identified thioamide pharmacophore. Binding affinities (K-d) were measured by surface plasmon resonance, revealing a modest improvement in potency over the initial 16 hits, with the best fragment found to bind to the apo enzyme with a K-d of 420 mu M, corresponding to a ligand efficiency of 1.8kJ/heavy atom. Four fragments identified represent useful starting points for the generation of leads that may ultimately be developed into new antimicrobial agents.