The human microbiota houses thousands of bacterial species working in a symbiotic relationship to achieve homeostasis. The rise of metagenomic sequencing of the complex microbiome has uncovered hundreds of links between the human microbiota and disease. Pathogenicity in the microbiota arise from an imbalance in microbial homeostasis known as dysbiosis. Chapter 1 discusses the positive and negative interactions between the microbiota and the human host with broad-spectrum antibiotic use being the major catalyst for dysbiosis. This chapter thoroughly examines various endeavors to develop pathogen specific, narrow-spectrum antimicrobials as alternative therapeutics for treatment of disease while maintaining host-microbiota homeostasis. Tilimycin is an enterotoxin produced by the opportunistic pathogen Klebsiella oxytoca that causes antibiotic associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway comprised of three proteins: NpsA, ThdA and NpsB. Chapter 2 reports the functional and structural characterization of the fully reconstituted NRPS system and reports the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was also confirmed through DNA adductomics techniques, detecting the putative N-2 guanine alkylation (dG-tilimycin) after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in vivo. Finally, we report the rational design of nonmicrobicidal small-molecule inhibitors that block tilimycin biosynthesis in whole cell K. oxytoca (IC50 = 28.6 ± 3.8 µM) through the inhibition of NpsA (KD = 29.2 ± 3.5 nM). Although the lung microbiota contains 106-fold less bacteria than the GI tract, it can house the deadliest pathogens of the microbiota including Klebsiella pneumoniae and Mycobacterium tuberculosis (Mtb). Hypervirulent Klebsiella pneumoniae (hvKP) is a life-threatening opportunistic pathogen that utilizes the iron assimilating siderophore, aerobactin. Aerobactin is an NRPS independent siderophore (NIS) that when genetically knocked out, results in significant attenuation of virulence. Similarly, Mtb utilizes the virulence factor mycolic acid to bolster its cell envelop, allowing innate resistance to antimicrobial treatment and immune response evasion. Chapter 3 investigates these two virulence factors produced by the lung pathogens Klebsiella pneumoniae and Mycobacterium tuberculosis. We elucidate the mechanistic features of IucC and IucA, the final synthetases of aerobactin biosynthesis. Stereoselective turnover of the penultimate substrate citryl-ahLys was unambiguously determined to be the (3S, 2'S) diastereomer using synthesized authentic standards. Mtb utilizes the fatty acid-AMP ligase, FadD32, to catalyzes the final step of mycolic acid biosynthesis, a critical virulence factor that bolsters the bacterium’s defense during infection of the lung. We rationally designed a small-molecule bisubstrate mimic of FadD32 which was shown to potently inhibit the enzyme (IC50 = 7.8 ± 1.2 µM). Aided by this tight binding inhibitor, the first FadD32 cocrystal structure was solved with high resolution, providing further insight into selective therapeutic design.