Ecosystems consist of primary producers, invertebrate and vertebrate consumers, and predators. Populations of each are regulated by direct and indirect interactions, as well as by parasites, pathogens, and microbiomes (bacterial and fungal communities both internal and external). These connections are also modulated by the local physicochemical environments, including water temperature, pH, and oxygen. To explore these connections at the whole ecosystem level, we collected eDNA water samples from 26 mainstem Colorado River sites and 22 tributary sites over the course of 5 years, with sampling occurring during multiple seasons. We also collected fecal samples over 400 fish, primarily native humpback chub and flannelmouth sucker, and nonnative rainbow trout. Invertebrate metabarcoding (COI) revealed 673 aquatic-associated insect and invertebrate taxa across the ecosystem, a major increase from previous reports, with major diversity partitions occurring between tributaries and the mainstem river, and across tributaries. General metazoan barcode primers (18S rRNA) revealed 115 parasite taxa, with strong parasite community differences across fish species, as well as differences in diet (differences in number of taxa of algae, plants, diatoms, and invertebrates consumed). Microbiome analysis (16S) identified distinct community types among fish species as well as gradients along the river continuum. Analysis of complex ecosystem data is a nontrivial task, with the generation of massive taxon co-occurrence matrices over time and space. Although Mantel tests identified connections between spatial location along the river and some diversity patterns (including a down-river shift in parasite, invertebrate, and microbiome communities), as well as temperature-related distribution patterns for some parasite species, causal connections generating distributions remain challenging. We are exploring the use of causal pathway analysis for identifying hidden or indirect causal connections based on co-occurrence data.