Freshwater ecosystems are under new and increasing threats from anthropogenic change. Ability to detect and predict consequences of environmental perturbations on ecosystem function and water quality is limited by the lack of empirical data over relevant time scales. Paleoecological records present a unique opportunity to broaden understanding of ecological transitions over decadal to millennial timescales. This study tested the occurrence of regime shifts to track changes throughout the lake food web beyond the typical instrumental era, using both “traditional” paleoecological proxies (e.g., cladoceran zooplankton, zoobenthos, and pigments) and more recently developed molecular genetic methods based on sedimentary DNA. We used sediment cores from the perialpine Lake Joux (Swiss Jura), where the history of human settlement and land-use practices in the catchment has been well documented since the Medieval period. Paleoecological evidence revealed an abrupt and unprecedented biological reorganization in the second half of the 20th century, following several centuries of relatively stable communities despite growing human pressure. Time-varying autoregression computed using dynamic linear modelling identified this transition, triggered by the onset of rapid cultural eutrophication in the 1950s, as a true regime shift. Since this time, despite decades of re-oligotrophication, biotic communities of Lake Joux have not returned to pre-disturbance composition, most likely due to other confounding factors, including climate warming, that may prevent the lake from returning to an earlier equilibrium state. Paleoecological reconstruction further suggested that cladocerans responded earlier to disturbance, which is highly relevant for lake monitoring and management strategies.