The microbiome of built structures has considerable influence over an inhabitant’s well-being, yet the vast majority of research has focused on human-built structures. Ants are well-known architects, capable of constructing elaborate dwellings, the microbiome of which is underexplored. Here, we explore the bacterial and fungal microbiomes in functionally distinct chambers within and outside the nests of Azteca alfari ants in Cecropia peltata trees. We predicted that A. alfari colonies 1) maintain distinct microbiomes within their nests compared to the surrounding environment, 2) maintain distinct microbiomes among nest chambers used for different functions, and 3) limit both ant and plant pathogens inside their nests. In support of these predictions, we found that internal and external nest sampling locations had distinct microbial communities, and A. alfari maintained lower bacterial richness in their “nurseries.” While putative animal pathogens were suppressed in chambers that ants actively inhabited, putative plant pathogens were not, which does not support our hypothesis that A. alfari defends its host trees against microbial antagonists. Our results show that ants influence microbial communities inside their nests similar to studies on human homes. Unlike humans, ants limit the bacteria in their nurseries and potentially prevent the buildup of insect-infecting pathogens. These results highlight the importance of documenting how indoor microbiomes differ among species, which might improve our understanding of how to promote indoor health in human dwellings.

adiv_Cecropia_Readinfile_Bact_OTUS_even1344alpha diversity values for 16S Bacterial datasetCecropia_Readinfile_fung_OTUS_even6130Raw sequence counts for ITS Fungal datasetCecropia_Readinfile_Bact_OTUS_even1344Raw sequence counts for 16S Bacterial dataset.adiv_Cecropia_Readinfile_fung_OTUS_even6130Alpha diversity values for ITS fungal datasetFunGuildIn.guildsFunGuild output of matched and unmatched sequences