This work aims to study the biophysical interactions of rifampicin (RIF) with three-dimensional macrophage membrane models, under environments with physiological and pathological relevance. The interaction of RIF with liposomes formed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in different pH values (i.e., 5.0, 6.2 and 7.4) was investigated by several biophysical techniques. The RIF's membrane concentration was quantified by partition coefficient (Kp) using derivative spectrophotometry. To predict the drug's location across the membrane, fluorescence quenching studies were performed using liposomes labeled with two different fluorescence probes. The effect of the drug on the biophysical parameters of the membrane was carried out by dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS). The overall results confirm that the interactions of RIF with membranes are pH-dependent, being much more pronounced at the acidic pH. A correlation between the effect of RIF on the biophysical properties of the membranes and the pH was found, which may be useful in the development of novel analogs with higher efficacy and fewer side effects, and also to understand the higher selectivity of RIF to the membranes of the infected cells, as well as its side effects.