Tuberculosis is one of the most relevant problems for global health care. The design of new drugs against tuberculosis is aimed at maximizing impact against the disease, as well as minimizing the toxicological effect on the lung surfactant. In this work, the antituberculosis drug Rifabutin is studied in combination with phospholipid Langmuir monolayers as models of the lung surfactant monolayer. The zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the anionic 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) were used as model phospholipids. A combination of in situ experimental techniques of Brewster angle microscopy, polarization-modulated infrared reflection-absorption spectroscopy, and UV-vis reflection spectroscopy with computer simulations has been used. The interactions between Rifabutin and the DPPC and DPPG Langmuir monolayers were described as the formation of an inclusion complex. The phospholipid-Rifabutin inclusion complex prevents the penetration of the Rifabutin into the alkyl chain region of the phospholipids, leading to a disruption of the monolayer structure and a possible toxicological effect.