Cross-kingdom interactions with plants were frequently related to microbial pathogens and herbivores. Yet, mutualistic interactions that involve multiple partners can confer cross-kingdom functional benefits, which have been understudied. Ant gardens (AGs) are recognized as one of the most sophisticated of all symbioses between ants and flowering plants, forming good models to study cross-kingdom interactions. The aim of this study was to examine whether ant-plant interactions can influence the community composition of root-associated fungi. We assessed whether two AG ant species, Camponotus femoratus and Neoponera goeldii, confer different physico-chemical properties to their nests, and affect root fungal community composition and fungal functional guilds in the bromeliad Aechmea mertensii. The diversity and community composition of root-associated fungi depended on ant species identity. The two ants had a contrasting influence on the structure and chemistry of the nest, and on the floristic diversity of the AGs. Multiple drivers may therefore determine the root-associated fungal communities. As the outcome of the ant-bromeliad interaction depends on the ant species, and because the plants are also involved in interactions with root-associated symbionts, this study provided evidence that ecologically relevant symbioses can be mediated by cross-kingdom interactions.