Understanding the factors shaping the diversity, composition, and function of wildlife gut microbiomes provides insight into host-microbe symbioses and can be used to improve our understanding of host ecology, evolution, and conservation. Previous studies examining wild fish gut microbiomes have identified habitat as a primary determinant, yet habitat effects can be confounded by food availability, environmental microbes, and local environmental conditions. To overcome these limitations, we investigated the factors driving variation in the gut microbiomes in 15 sympatric fish species using 16S rRNA gene sequencing for gut and environmental microbe profiling as well as multi-locus metabarcoding (18S, COI, 12S) for comprehensive diet characterization. Our results revealed that microbial diversity is highest in environmental biofilms and lowest in water samples, while gut microbiome diversity is highly variable among species of freshwater fishes. Comparative community composition analyses highlighted significant differentiation between environmental and gut microbiome samples, indicating a filtering process whereby most environmental microbes do not persist in the gut microbiome. When considering host diet, herbivorous species displayed the highest gut microbiome alpha diversity but lowest beta diversity dispersion, while predators and omnivores exhibited lower alpha diversity and higher dispersion. Diet diversity (aggregated across three loci) and phylogenetic distance among species made significant contributions to gut microbiome structure, underscoring the combined impacts of ecological and evolutionary processes in shaping fish gut microbial communities. These findings provide novel insights into the factors governing gut microbiome diversity in sympatric fish species and provide a foundation for understanding mechanisms driving microbial community assembly and dynamics in aquatic ecosystems.