Infection by pathogenic mycobacteria such as Mycobacterium tuberculosis disrupts the membrane of the Mycobacterium-Containing Vacuole (MCV). The key effector EsxA, secreted via the ESX-1 type-VII system, is pivotal in this process, yet its membranolytic activity is not fully elucidated. Infecting the amoeba Dictyostelium discoideum with Mycobacterium marinum , we demonstrate that the composition of the MCV membrane, notably its sterol-rich microdomains, significantly influences damage and rupture. Disruption of these microdomains through the knockout of organizing proteins, termed vacuolins, or through sterol depletion, markedly diminishes M. marinum -induced membrane damage and cytosolic escape, thereby increasing cellular resistance to infection. Furthermore, we establish that vacuolins and sterols are essential for the in vitro partitioning of EsxA within membranes. Extending our findings to mammalian cells, we show that the role of microdomain organizers and sterols is evolutionarily conserved; specifically, flotillin knockdown and sterol depletion enhance the resistance of murine microglial cells to M. marinum infection. Our results underscore the critical role of host membrane microdomains in facilitating mycobacterial membranolytic activity and subsequent cytosolic access, pivotal for a successful infection.