Symbioses between plants and fungi, fungi and ants, and ants and plants all play important roles in ecosystems. Symbioses involving all three partners appear to be rare. Here, we describe a novel tripartite symbiosis in which ants and a fungus inhabit domatia of an ant-plant, and present evidence that such interactions are widespread. We investigated 139 individuals of the African ant-plant Leonardoxa africana for occurrence of fungus. Behaviour of mutualist ants toward the fungus within domatia was observed using a video camera fitted with an endoscope. Fungi were identified by sequencing a fragment of their ribosomal DNA. Fungi were always present in domatia occupied by mutualist ants but never in domatia occupied by opportunistic or parasitic ants. Ants appear to favour the propagation, removal and maintenance of the fungus. Similar fungi were associated with other ant-plants in Cameroon. All belong to the ascomycete order Chaetothyriales; those from L. africana formed a monophyletic clade. These new plant-ant-fungus associations seem to be specific, as demonstrated within Leonardoxa and as suggested by fungal phyletic identities. Such tripartite associations are widespread in African ant-plants but have long been overlooked. Taking fungal partners into account will greatly enhance our understanding of symbiotic ant-plant mutualisms.