Isoniazid (INH) is one of the primary drugs used in tuberculosis treatment and its encapsulation in liposomal vesicles can both improve its therapeutic index and minimize toxicity. Here we consider mixtures of hydrogenated soy phosphatidylcholine-phosphatidylglycerol (HSPC-DPPG) to get novel biocompatible liposomes for INH delivery. We determined INH encapsulation efficiency by coupling for the first time UV and Laser Transmission Spectroscopy and we showed that HSPC-DPPG liposomes can load more INH than expected from simple geometrical arguments, thus suggesting the presence of drug-lipid association. To focus on this aspect, which has never been explored in liposomal formulations, we employed several complementary techniques, such as dynamic and static light scattering, calorimetry and surface pressure measurements on lipid monolayers. We find that INH-lipid interaction increases the entrapment capability of liposomes due to INH adsorption. Moreover, the preferential INH-HSPC dipole-dipole interaction promotes the modification of lipid ordering, favoring the formation of HSPC-richer domains in excess of DPPG. Our findings highlight how investigating the fundamental aspects of drug-lipid interactions is of paramount importance for the optimal design of liposomal nanocarriers.