<p>Studying host-associated microbiome assembly is key to understanding microbial and host evolution and health. While honey bee microbiome have been a central model for such investigations among pollinators, they overlook the diversity of eusocial dynamics and multi-kingdom interactions. Stingless bees—a diverse group of highly eusocial insects that includes managed species, varies in colony biology, and harbors a symbiotic yeast essential for larval development in at least one species—offer a valuable complementary system to study microbiome assembly under an eco-evolutionary context. Using amplicon sequencing, metagenomics, and microbial experiments, we investigate the drivers of microbiome assembly in stingless bee colonies. We reveal a spatially structured, site-adapted microbiome, where high microbial influx hive components are segregated from the brood, which harbors a stable, multi-kingdom community. We show that the brood microbiome is not only physically protected but also maintained through selective bacterial-fungal interactions and abiotic conditions shaped by bees and their symbionts, such as temperature and pH. Our findings uncover multi-layered mechanisms shaping eusocial superorganism microbiomes, from host biology to cross-kingdom interactions, while providing critical insights into microbiome maintenance of important pollinators.</p>