Chitosan has been reported to inhibit spore germination and mycelial growth in plant pathogens, but its mode of antifungal action is poorly understood. Following chitosan treatment, we characterized plasma membrane permeabilization, and cell death and lysis in the experimental model, Neurospora crassa. Rhodamine-labeled chitosan was used to show that chitosan is internalized by fungal cells. Cell viability stains and the calcium reporter, aequorin, were used to monitor plasma membrane permeabilization and cell death. Chitosan permeabilization of the fungal plasma membrane and its uptake into fungal cells was found to be energy dependent but not to involve endocytosis. Different cell types (conidia, germ tubes and vegetative hyphae) exhibited differential sensitivity to chitosan with ungerminated conidia being the most sensitive.