The ability of Mycobacterium tuberculosis to remain dormant after primary infection represents the prime cause of new TB cases throughout the world. Hence, diagnosis and treatment of individuals hosting dormant mycobacterium is one of the crucial strategies to be adopted for the prevention of Tuberculosis. Among many strategies unleashed by the latent bacterium, one of them is scavenging host cholesterol for carbon source. Cholesterol modifies lipid membranes over many scales and here, its effect on mycobacterial membrane biophysics and the subsequent effect on partitioning of antibiotics into cholesterol- enriched mycobacterial membranes was investigated. Our research showed that cholesterol alters the phase state behavior of mycobacterial outer membrane lipids by enhancing the overall membrane order at the headgroup and acyl chain region and is integrated into both ordered and disordered domains/phases, with a preference for the latter. Exogenous cholesterol further alters the drug partitioning behavior of structurally different drugs, pointing to a larger clinical potential of using more hydrophobic medications to target dormant bacteria.