Fungus-growing ants (Attini) have evolved an obligate dependency upon a basidiomycete fungus that they cultivate as their food. Less well known is that the crop fungus is also used by many attine species to cover their eggs, larvae and pupae. The adaptive functional significance of this brood covering is poorly understood. One hypothesis to account for this behaviour is that it is part of the pathogen protection portfolio when many thousands of sister workers live in close proximity and larvae and pupae are not protected by cells, as in bees and wasps, and are immobile. We performed behavioural observations on brood covering in the leaf-cutting ant Acromyrmex echinatior, and we experimentally manipulated mycelial cover on pupae and exposed them to the entomopathogenic fungus Metarhizium brunneum to test for a role in pathogen resistance. Our results show that active mycelial brood covering by workers is a behaviourally plastic trait that varies temporally, and across life stages and castes. The presence of a fungal cover on the pupae reduced the rate at which conidia appeared and the percentage of pupal surface that produced pathogen spores, compared to pupae that had fungal cover experimentally removed or naturally had no mycelial cover. Infected pupae with mycelium had higher survival rates than infected pupae without the cover, although this depended upon the time at which adult sister workers were allowed to interact with pupae. Finally, workers employed higher rates of metapleural gland grooming to infected pupae without mycelium than to infected pupae with mycelium. Our results imply that mycelial brood covering may play a significant role in suppressing the growth and subsequent spread of disease, thus adding a novel layer of protection to their defence portfolio.