Resistance to existing antimicrobial agents is a global threat to human health care, and new drugs with novel mechanisms of action are required in order to keep pace with the emergence of drug resistant pathogens. The bacterial fatty acid biosynthesis (FAS-II) and menaquinone pathways represent attractive yet relatively unexploited targets for new antibiotic development and consequently there is significant interest in developing potent inhibitors of enzymes in these two pathways. My research is mainly focused on the mechanism and inhibition of the enoyl-ACP reductases from the FAS-II pathway and dihydroxynaphthoyl-CoA synthase from the menaquinone pathway. The mechanism and inhibition of the enoyl-ACP reductase from Mycobacterium tuberculosis (InhA) was studied. A series of compounds with nanomolar affinity for the enzyme and reduced lipophilicity were identified. In addition, a slow-onset inhibitor of InhA that has a K<sub>1</sub> value of 22 pM and a residence time of 23 min on the enzyme was identified and characterized. Site-directed mutagenesis and X-ray structural studies demonstrated that slow onset inhibition of InhA results in ordering of a substrate-binding loop that covers the entrance to the binding pocket and thereby locks the inhibitor in the substrate binding cavity and increases its residence time. This is significant since long drug-target residence time is thought to be an important factor for in vivo drug activity. Studies of enoyl-ACP reductases have also been extended to Burkholderia pseudomallei (bpmFabIs) and Yersinia pestis (ypFabV). Mechanistic studies were performed on two FabI homologues in B. pseudomallei, which demonstrated that only one (bpmFabI-1) has enoyl-ACP reductase activity. As a prelude to rational inhibitor development, the sensitivity of bpmFabI to four diphenyl ethers has been evaluated. In each case the compounds are nanomolar slow onset inhibitors. Reduction in MIC values is observed for the Burkholderia sp. efflux pump mutant with all diphenyl ethers suggesting that bpmFabI-1 is a suitable target for drug discovery provided that efflux can be circumvented. Mechanistic studies are also performed on the enoyl ACP reductase FabV homologue in Y. pestis. Steady-state kinetics has been used to study the reaction mechanism of ypFabV. Preliminary inhibition studies indicate that diphenyl ethers are not promising leads for developing potent FabV inhibitors. Interestingly, during the study of the FabI in B. pseudomallei, we found this organism has a high level of unsaturated fatty acids (UFAs), but lacks fabA and fabB homologues that normally found in the UFA biosynthesis pathway. We attempted to identify the putative trans-2, cis-3-enoyl-ACP isomerase (FabM), which is a key enzyme in alternative UFA biosynthesis pathway. However, the candidate was characterized and found as a trans-2, cis-3-enoyl-CoA isomerase. Through sequence alignment with other members of the crotonase superfamily, conserved catalytic residues were recognized and enzymatic mechanism for this candidate was proposed. Finally, the mechanism and inhibition of the dihydroxynaphthoyl-CoA synthase (MenB) from Mycobacterium tuberculosis was studied. In the menaquinone pathway, MenB catalyzes the formation of a carbon-carbon bond through a Dieckmann condensation. We compared the mechanism of the M. tuberculosis MenB enzyme with that of MenB from E. coli. Kinetic data and X-ray crystallography suggest that MenBs from M. tuberculosis and E. coli utilize the same substrate for catalysis and share the same reaction mechanism. In addition, we performed an structure activity relationship study on MenB inhibitors, based on two studied leads: the 2-amino-4-oxo-phenylbutanoic acids and the benzoxazinones from high throughput screen. The most potent compound against MenB exhibits excellent inhibition in vitro with the K<sub>1</sub> value of 18 nM. These studies will help us to validate MenB as a target for the development of novel microbial chemotherapeutics.