Global biodiversity is threatened by the anthropogenic restructuring of animal communities, rewiring species interaction networks in real-time as individual species are extirpated or introduced. Biodiversity and functionality are not only declining, but human-induced hybridization of wildlife and the shuffling of biomes are also becoming more common. Conservation science and adaptive ecosystem management demand more rapid, quantitative, and non-invasive technologies for robustly capturing changing biodiversity, quantifying species interactions, and measuring ecosystem function to protect remaining systems. Using both non-invasive DNA metabarcoding of diet DNA (dDNA) and network theory, the aim of this dissertation was to develop molecular ecological network analysis (MENA) as an ecosystem assessment tool to address these needs. These methods were first tested in a well-studied biological preserve (Jasper Ridge Biological Preserve, Stanford, CA), where they more rapidly, accurately, and effectively captured the broader biodiversity of the area in comparison to other methodologies (camera trap and soil eDNA), while reconstructing and unveiling the hidden complexity in trophic structure and interaction networks within the community. I then applied MENA in a hybrid ecotone in Garamba National Park, Democratic Republic of Congo to understand the consequences of two African elephant species hybridizing, the critically endangered Forest (Loxodonta cyclotis) and endangered Savanna elephant (L. africana). The aim was to determine how hybrid introgression may currently be shaping the ecology (diet selection and habitat use) and health of the individual elephant (gut endoparasites), while assessing a suite of demographic (age, sex), environmental (habitat type), and social (family vs bull groups) characteristics. A third of this population are hybrids and the admixed genetics influenced helminth composition and significantly reduced host-specific parasite infection. Garamba forest elephants and hybrid elephants have very similar ecologies (dietary composition and habitat use), however, this population of elephants is unique in diet and habitat use compared to other typically frugivorous forest elephant populations and might be fulfilling a vacant niche within the grasslands, maintaining ecosystem function. This research will hopefully equip conservation ecologists and managers with a powerful tool to measure biodiversity and species interactions to assess the integration and impact of novel species in the ecosystem, unveil hidden multitrophic interactions and community structure, and identify key and vulnerable species within a terrestrial system. [link to publication]