Very few FabF or FabB ligands that are not derived from one of the known natural product inhibitors (Figure ) are known. Using phenotypic screening, an inhibitor of C. trachomatis FabF has been discovered, however, the binding mode remains unclear. We have discovered a PaFabF ligand using virtual screening but optimization has proven difficult. Further, indazole sulphonamides have been reported as inhibitors of M. tuberculosis KasA (an ortholog of FabB). Finally, two B. melitensis FabB crystal structures bound to fragments have been published. Crystal structures of FabF and FabB from various organisms have been determined and thus a structurebased approach can be used for the discovery of new inhibitors. Several of the known structures are in complex with a fatty acid, but none is in complex with malonyl-ACP. However, in an effort to further elucidate the reaction mechanism, structures with cross-linked substrate mimetic-bearing ACPs have been determined. The C12 fatty acid part of the C12-substrate mimetic overlaps well with the fatty acid part in dodecanoyl-FabB (PDB ID 1EK4, not shown ) and cerulenin bound to FabB while the activated malonyl mimicking part is located in the same pocket as the inhibitor platensimycin (Figure ). Consequently, both the fatty acid pocket and the malonyl-CoA binding site can be targeted for drug discovery. In an effort to further explore the chemical space of FabF ligands, we have carried out fragment screening by X-ray crystallography making use of the XChem facility at the Diamond light source. We opted to use an intermediate state mimicking protein variant for the screening, as the most potent ligand known to date, platensimycin, binds to this state. In this state, the fatty acid binding site is still accessible for ligands, so both pockets can be screened simultaneously. Screening has resulted in 17 hits binding in either of the substrate binding sites or a channel in the dimer interface connection these pockets in addition to a further 31 hits binding at other positions. Here, we report on this fragment screening campaign as well as on our attempts to further explore selected hits. The presented work forms a comprehensive basis upon on which more diverse and potent FabF inhibitors can be developed in the future.