The attine ant-microbe association is a complex symbiosis in which ants cultivate basidiomycete fungi as their food source. Like humans, attine ants use several strategies to protect themselves and their cultivar from pathogens that may enter a nest either via the vegetable substrate collected by the ants for fungal cultivation or via arthropod vectors (e.g., mites, collembola). Fungal garden pathogens have been called “weeds” but, with the exception of the garden parasite Escovopsis, the roles of the many other filamentous microfungi in attine gardens have not been well characterized. Some microfungal species can be found at significant frequencies in attine gardens, raising the question whether these microorganisms are specialized pathogens, general contaminants, or additional symbionts performing an adaptive role in gardens. As a first step towards addressing these questions, we surveyed the microfungi distribution in Acromyrmex spp. nests in southern Brazil with molecular phylogenetic techniques. A total of 34 fungal taxa were isolated from 41 Acromyrmex spp. nests (11 ant species). The most frequent species were: Fusarium oxysporum (41 %), Escovopsis weberi (24 %) and Cunninghamella binariae (17 %). In a phylogenetic analysis, F. oxysporum isolates clustered with other F. oxysporum strains with known environmental origins (soil and leaves). Some C. binariae isolates formed a group composed only by attine ants’ isolates. Moreover, E. weberi isolates clustered with Escovopsis strains reported previously from other leaf-cutting ants species from Central America, and no clear biogeographic separation in Central and South American Escovopsis lineages was evident. The rich assemblage of filamentous fungi in attine gardens shows that ants and their cultivars are in contact with diverse microfungi that are commonly found in the environment. To elucidate the generality of the findings, the survey of microfungi from South Brazilian Acromyrmex gardens will be compared with an ongoing survey in North American attine gardens.