To determine the effects of cytochalasin E, isolated from the extremophile fungus Aspergillus felis, on the cells of Paracoccidioides brasiliensis Pb18. Cytochalasin E showed a minimal inhibitory concentration of 3·6μmoll-1 and minimum fungicidal concentration of 7·2μmoll-1 on P. brasiliensis by invitro microdilution and IC50 >964·0μmoll-1 on murine macrophages. Its selectivity index (>263) indicated that this compound has selectivity for fungal cells. Morphological alterations were determined by optical and fluorescence microscopy, as well as scanning and transmission electron microscopy. Cytochalasin E affected P. brasiliensis bud-forming pseudohyphae, cell morphology, cell walls and cell membranes; caused the release of cellular material; and resulted in the production of reactive oxygen species. In murine macrophages, it affected cytoskeletal actin and inhibited phagocytosis. Cytochalasin E may be useful as an antifungal prototype against P. brasiliensis and in studies on phagocytosis. Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM). Treatment is prolonged to control the clinical manifestations and prevent relapse. The study on the effects of cytochalasin E in P. brasiliensis is important because it can be used as a prototype for new antifungal drugs and consequently, broadens the treatment options for PCM.