Honey bees are a model for host–microbial interactions with experimental designs evolving towards conventionalized worker bees. Research on gut microbiome transmission and assembly has examined only a fraction of factors associated with the colony and hive environment. Here, we studied the effects of diet and social isolation on tissue-specific bacterial and fungal colonization of the midgut and two key hindgut regions. We found that both treatment factors significantly influenced early hindgut colonization explaining similar proportions of microbiome variation. In agreement with previous work, social interaction with older workers was unnecessary for core hindgut bacterial transmission. Exposure to natural eclosion and fresh stored pollen resulted in gut bacterial communities that were taxonomically and structurally equivalent to those produced in the natural colony setting. Stressed diets of no pollen or autoclaved pollen in social isolation resulted in decreased fungal abundance and bacterial diversity, and atypical microbiome structure and tissue-specific variation of functionally important core bacteria. Without exposure to the active hive environment, the abundance and strain diversity of keystone ileum speciesGilliamella apicolawas markedly reduced. These changes were associated with significantly larger ileum microbiotas suggesting that extended exposure to the active hive environment plays an antibiotic role in hindgut microbiome establishment. We conclude that core hindgut microbiome transmission is facultative horizontal with 5 of 6 core hindgut species readily acquired from the built hive structure and natural diet. Our findings contribute novel insights into factors influencing assembly and maintenance of honey bee gut microbiota and facilitate future experimental designs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00248-022-02025-5.