The human gut hosts diverse microbes essential for metabolism, immune function, and disease prevention. Imbalance, or dysbiosis, of the gut microbiota contributes to various diseases, with diet exerting a stronger influence than genetics. We investigated the effects of five diets, chow, high-carbohydrate, high-fat, high-protein, and high-fiber, on the gut microbiota (bacteria) and mycobiota (fungi) in conventionally housed mice, highlighting implications for pharmaceutical research. Gut microbial composition varied with diet: the phylum Bacillota dominated in the chow, high-carbohydrate, and high-fat diet groups, whereas Bacteroidota predominated in the high-protein and high-fiber diet groups. Linear Discriminant Analysis Effect Size analysis identified distinct bacterial taxa enriched in each dietary group compared with chow. Fungal analysis showed a shift from Ascomycota dominance in chow to Basidiomycota in the other diet groups. Correlation analysis indicated that bacterial–fungal relationships varied with diet; notably, the high-protein diet group showed numerous strong negative correlations between bacterial and fungal families. These findings emphasize that diet shapes microbial composition and interactions in animal models, providing baseline information for considering diet-associated microbial variation in preclinical research.