Aspergillus fumigatus and free-living amoebae are common inhabitants of soil. Mechanisms of A. fumigatus to circumvent the amoeba's digestion may facilitate overcoming the vertebrate macrophage defence mechanisms. We performed co-culture experiments using A. fumigatus conidia and the amoeba Acanthamoeba castellanii. Approximately 25% of the amoebae ingested A. fumigatus conidia after 1 h of contact. During intra-amoebal passage, part of the ingested conidia was able to escape the food vacuole and to germinate inside the cytoplasm of A. castellanii. Fungal release into the extra-protozoan environment by exocytosis of conidia or by germination was observed with light and transmission electron microscopy. These processes resulted in structural changes in A. castellanii, leading to amoebal permeabilization without cell lysis. In conclusion, A. castellanii internalizes A. fumigatus conidia, resulting in fungal intracellular germination and subsequent amoebal death. As such, this interaction highly resembles that of A. fumigatus with mammalian and avian macrophages. This suggests that A. fumigatus virulence mechanisms to evade macrophage killing may be acquired by co-evolutionary interactions among A. fumigatus and environmental amoebae.