In recent decades, interest has grown in understanding how pre-industrial activities have contributed to trace metals pollution into the environment at the local and regional scales. Southern Tuscany hosts some of the most important metallogenic provinces in Italy exploited for almost the last three millennia. Studying the history of trace metals pollution in this area offers insights into the temporal and spatial scope of human-environment interactions, evaluates the severity of pollution, and can trace the local natural background values. To explore these aspects, trace metals, major elements, and lead (Pb) isotope ratios were analyzed in an 8000-year sediment records from Lake Accesa, a karst lake located on the southern border of the Colline Metallifere mining district. The findings indicate that Pb in Lake Accesa is mainly related to sulfide polymetallic deposits that surround the lake catchment. The first signal of Pb pollution dates to about 3300 BCE (Before Common Era) during the Copper Age and it is consistent with the archaeological evidence of Southern Tuscany. Additional human-induced Pb pollution signals can be observed in the Bronze Age (∼1550 BCE), and a long phase beginning in the Middle Ages (from ∼700 CE[Common Era]). Between 1000 and 1700 CE, Pb reached the highest concentrations, corroborating the intensity of mining activity during and after the Medieval period. These findings reveal that pre-industrial activities left a significant legacy of potential toxic elements in the environment, resulting in pollution levels that exceed those related to recent activities associated with the Anthropocene. The Lake Accesa record further indicates that mining of sulfide deposits in the Etruscan period was minimal and even completely negligible during Roman times, probably due to the exploitation of other ore deposits.