Antitubercular therapy has been successful in bringing down the death rate due to tuberculosis. However, there are cases where patients survive with persistent Mycobacterium at the end of their treatment cycles, demanding for new markers or agents to treat drug resistant variants^1,2. Highly complex mycobacterial membrane envelope with outer and inner membrane layers that possess distinct compositions and membrane properties at different growth stages, act a barrier to therapeutic agents, attenuating their intracellular concentrations and hence leading to drug resistance³. Australian frog peptides aurein1.2 and maculatin1.1 have membrane-disruptive properties^4,5 which could be exploited to enhance the antibiotic uptake in Mycobacterium. In this work, a combination of biophysical techniques was used to study the lipid-specific peptide interaction patterns on mycobacterial cell envelope layers. Microscale thermophoresis studies assured specific binding of the peptides to Mycobacterium smegmatis (Msm) at or above ~2µM, where concentration dependent cell-size reduction was observed upon aurein interaction. Surface plasmon resonance and atomic force microscopy studies revealed that these peptides interact with the lipid membrane to induce membrane disruption by either pore formation or lipid clustering. This mechanism induced enhanced permeability of the lipid membranes, which when cross-checked on whole Msm cell, assured the absence of any non-lipid component (peptidoglycan or lipoarabinomannan) in initiating the membrane permeability. This expanded the application of these peptides in combination drug therapy, where when used with small anti-tubercular drugs such as amikacin, these peptides spur up the drug uptake to enhance intracellular drug concentrations and induce improved cytotoxicity up to 150%. Taken together, leveraging the membrane-drug interaction profiles unique to mycobacteria, membrane-disruptive peptides and anti-tubercular drugs may provide strategies for new combination TB therapies⁶.