Acyl-CoA thioesterases are a family of proteins which catalyse the cleavage of activated fatty Acyl-CoA molecules to release coenzyme A and the corresponding fatty acid. Research within bacterial species has demonstrated the presence of thioesterases crucial for the virulence of these pathogens. The characterisation of such proteins by x-ray crystallography and a range of complementary molecular techniques provide a platform for rational drug design against these pathogenic organisms. In 2011 a number of potential inhibitors for a related human thioesterase were investigated with the potential to reduce inflammation and form the basis for an anti-inflammatory drug (Forwood et al., 2007, Kirkby et al., 2010, Swarbrick et al., 2011, Serek et al., 2006, Kirkby et al., 2011). Building on this approach, we have characterised a number of thioesterase proteins within pathogenic bacteria as a basis for developing in vitro inhibitors and potential drug molecules. To date the cloning, expression and purification of a number of thioesterases from Y. pseudotuberculosis and L. pneumophila have been undertaken. Subsequent crystal trials have yielded diffraction quality crystals for two thioesterases, tesB from Y. pseudotuberculosis and an unnamed hypothetical thioesterase from L. pneumophila (L2), from which the structures of tesB (4GWH) and L2 have subsequently been solved. Functional data for tesB has revealed a broad range acyl-CoA thioesterase with high activity against decanoyl-CoA (C10). Furthermore the structure for ACOT12 has been solved and the activity against a range of substrates from acetyl CoA (C2) up to Arachidonoyl-CoA (C20:4) investigated reveal activity against the substrate hexanoyl-CoA (C6). These studies provide an enhanced understanding of the role of these thioesterases in prokaryotic and eukaryotic organisms, and a basis for development of inhibitors and drug design.