<p>Leaf-cutter ant species in the Attini tribe have evolved the ability to cultivate fungi for food, a crucial adaptation that has allowed them to become ecologically important herbivores but also insect pests throughout the Neotropics. While the primary fungal gardens of these insects have been extensively researched for decades, their associated bacterial communities have received comparatively less attention. This is especially true for <i>Atta mexicana</i>, a species distributed throughout Central and North America, whose fungus garden’s bacterial communities have never been investigated. Using a metataxonomic approach, we explored the assembly of the bacterial communities associated with the fungus garden of <i>Atta mexicana</i> maintained under controlled conditions. Our findings revealed differences in bacterial community assembly associated with the fungus garden’s strata level that differed between queens collected from distinct colonies. These results suggest ongoing ecological succession processes mainly driven by spatial specialization within the fungal garden and provide numerous avenues for future research.</p>