Mycobacterium tuberculosis (Mtb), the cause of the human pulmonary disease tuberculosis (TB), continues to be the leading cause of death by a single infectious agent. Although a treatment regimen has existed for over half a century, TB incidence remains high, in large part due to the length of treatment required for cure. During infection, Mtb must overcome numerous environmental challenges including nutrient deprivation and oxygen limitation. Mtb is uniquely adapted to long-term intracellular survival and thus bacterial clearance is difficult to achieve, even in the face of a robust immune response and a strict antibiotic treatment regimen.Identification and interrogation of genes and pathways that are critical for Mtb survival will likely aide in developing improved therapeutics for treating TB. In order to investigate Mtb gene essentiality, we conducted a genome-wide genetic screen using a CRISPRi knockdown library and an ex vivo challenge model. Many of the genes found to be essential are involved in various metabolic pathways including lipid catabolism and iron homeostasis. Individual knockdown strains were generated targeting three of the metabolic pathways identified in the genetic screen. Each of the knockdown strains led to a reduced bacterial burden in mouse lungs, confirming in vivo essentiality. Due to Mtb’s dependence on cholesterol as a carbon and energy source, we conducted a high-throughput screen of over 1.1 million compounds in order to identify novel inhibitors of Mtb cholesterol metabolism. One of the lead compounds identified in this screen, sAEL057, unveiled a previously unknown linkage between iron homeostasis and cholesterol catabolism in Mtb. Another compound, sAED182, leads to nitric oxide release and is able to kill persistent Mtb more effectively than current anti-TB drugs in vitro. Thus, our genetic screen identified pathways Mtb is most reliant on to maintain infection, and our chemical screen and subsequent characterization identified novel compounds capable of perturbing these metabolic pathways.