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Global biodiversity is threatened by the anthropogenic restructuring of animal communities, rewiring species interaction networks in real-time as individuals are extirpated or introduced. Conservation science and adaptive ecosystem management demand more rapid, quantitative, and non-invasive technologies for robustly capturing changing biodiversity and quantifying species interactions. Here we develop molecular ecological network analyses (MENA) as an ecosystem assessment tool to address these needs. To construct the ecological network, we used environmental DNA from feces to identify the plant and mammal diet of two carnivores: puma (Puma concolor) and bobcat (Lynx rufus); two omnivores: coyote (Canis latrans) and gray fox (Urocyon cinereoargenteus); and two herbivores: black-tailed deer (Odocoileus hemionus) and black-tailed jackrabbit (Lepus californicus) in a well-studied Californian reserve. To evaluate MENA as a comprehensive biodiversity tool, we applied our framework to identify the structure of the network, patterns of trophic interactions, key species, and to assess its utility in capturing the biodiversity of the area. The high dietary taxonomic resolution enabled the assessment of species diversity, niche breadth and overlap. The network analysis revealed a dense ecological network with a high diversity of weakly connected species and a community that is highly modular and non-nested. The significant prevalence of tri-trophic chain and exploitative competition patterns indicates (i) the removal or reintroduction of a top predator would trigger a trophic cascade within this community, directly affecting their prey and indirectly the plant communities, and (ii) the potential impact of indirect effects between two predators that consume the same prey. These results suggest that the recent resurgence of puma in the study area may impact the herbaceous and woody vegetation and the population size of other predators. This effect of fluctuating predator populations and plant communities could be predicted through MENA’s fine-scale assessment of the diet selection and the identified keystone species. Although just using a subset of species, MENA more rapidly, accurately, and effectively captured the broader biodiversity of the area in comparison to other methodologies. MENA reconstructed and unveiled the hidden complexity in trophic structure and interaction networks within the community, providing a promising toolkit for biodiversity and ecosystem management.