Histoplasma capsulatum is a dimorphic, thermal, and nutritional fungus. In the environment and at an average temperature of 28 °C, it develops as a mold that is composed of infecting particles. Once in the host or in cultures at 37 °C, it undergoes a transition into the parasitic form. In the present work, we performed chemical extraction and characterization using chromatography techniques of the associated lipid composition of the external surface of the cell wall of the mycelial phase of two isolates of the H. capsulatum: one clinical and one environmental. Several differences were evidenced in the fatty acids in the phospholipid composition. Surface pressure-area isotherms and compression module curves of the Amphotericin B and lipid extract monolayers, as well as (AmB)-lipid extract mixed monolayers were recorded. Results show a high affinity of AmB towards lipid extracts. The most stable monolayers were formed by AmB + environmental with a mass ratio of 1:3 and AmB + clinical with a mass ratio of 1:2. Knowledge of the AmB aggregation processes at a molecular level and the characterization of the lipid extracts allows the possibility to understand the interaction between the AmB and the lipid fractions of H. capsulatum.