We examine how biomolecular analyses of archaeological fauna can generate conservation-relevant ecological knowledge that is inaccessible from historical observations or modern monitoring alone. Through three late Holocene Lower Great Lakes case studies, we show how stable isotope, aDNA, and proteomic analyses clarify species ecology, ecosystem change, and extinction dynamics at temporal and spatial scales relevant to restoration and management. Analyses of Atlantic salmon from the Lake Ontario watershed demonstrate that individuals from this now-extinct population were freshwater-resident rather than anadromous, resolving long-standing uncertainty about life history and establishing critical baselines for reintroduction planning. Isotopic and proteomic analyses of salmon, lake trout, and whitefishes show that Lake Ontario’s nitrogen cycle and food web structure remained stable for centuries before undergoing an abrupt shift coincident with industrial-scale deforestation in the early nineteenth century, identifying forest clearance—rather than long-term Indigenous land use—as a threshold driver of watershed-scale nutrient disruption. Finally, isotopic and ancient DNA evidence from passenger pigeon demonstrates substantial dietary plasticity prior to extinction, challenging habitat-loss-only explanations and underscoring the dominant role of intensive exploitation. We conclude by outlining future research directions linking biomolecular archaeological datasets from endangered species with conservation planning, emphasizing sustained collaboration among archaeologists, ecologists, and Indigenous communities.