Mycobacterium tuberculosis utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. ESX-1 is one of the essential and well-studied secretion systems which transport various virulence factors, including EspB. EspB, a ~36 kDa secreted substrate, has been implicated to play vital role in protecting the bacteria from hostile environment within the host cell phagosome. It is also involved in bacterial pathogenesis and has been shown to bind phospholipids. Recently, two cryo-EM structures of EspB full-length and the secreted isoforms were resolved. Despite the availability of multiple high-resolution structures of EspB, the physiological relevance and mechanism of virulence of this secreted substrate remains poorly characterized. In this current work, we implemented cryo-EM-based structural studies, including various functional assays, TEM imaging, and biophysical approach to demonstrate the interaction of EspB with lipids and bio-membrane. Our findings also indicated that EspB may play a crucial role in binding to and rupturing host mitochondrial membrane. Through cryo-EM studies we were able to show the possible membrane-binding region of EspB. Our study sheds light on host-pathogen interactions and bacterial pathogenesis mediated by EspB.