N-geranyl-N΄-(2-adamantyl)ethane-1,2-diamine (SQ109) is a tuberculosis drug that has high potency againstMycobacterium tuberculosis (Mtb)and may function by blocking cell wall biosynthesis. After the crystal structure of MmpL3 fromMycobacterium smegmatisin complex with SQ109 became available, it was suggested that SQ109 inhibits Mmpl3 mycolic acid transporter. Here, we showed using molecular dynamics (MD) simulations that the binding profile of nine SQ109 analogs with inhibitory potency against Mtb and alkyl or aryl adducts at C-2 or C-1 adamantyl carbon to MmpL3 was consistent with the X-ray structure of MmpL3 – SQ109 complex. We showed that rotation of SQ109 around carbon–carbon bond in the monoprotonated ethylenediamine unit favors twogaucheconformations as minima in water and lipophilic solvent using DFT calculations as well as inside the transporter’s binding area using MD simulations. The binding assays in micelles suggested that the binding affinity of the SQ109 analogs was increased for the larger, more hydrophobic adducts, which was consistent with our results from MD simulations of the SQ109 analogues suggesting that sizeable C-2 adamantyl adducts of SQ109 can fill a lipophilic region between Y257, Y646, F260 and F649 in MmpL3. This was confirmed quantitatively by our calculations of the relative binding free energies using the thermodynamic integration coupled with MD simulations method with a mean assigned error of 0.74 kcal mol−1compared to the experimental values.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s10822-023-00504-6.