An analysis of environmental DNA (eDNA) was used to document the fish community in the watershed managed by the Organisme de bassin versant de la zone du Chêne and in the Forêt de la Seigneurie de Lotbinière (FSL) (Québec, Canada). The main goal was to document species diversity and relative abundance at different sites within the FSL and in the adjacent agroforestry landscape, and to compare the species richness detected by eDNA with that obtained during previous traditional inventories. Forty-one species of fish were detected, including 2 species sought after by anglers, the brook charr (Salvelinus fontinalis) and smallmouth bass (Micropterus dolomieu), and 2 species of particular conservation interest, the channel darter (Percina copelandi) and cutlip minnow (Exoglossum maxillingua). Four species were new records for the watershed. When fish do not need to be caught, eDNA sampling offers a highly effective and much less invasive means of obtaining a detailed picture of the species present. Furthermore, it requires a lower sampling effort than traditional survey techniques.