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Although rare, temporally and taxonomically highly-resolved palaeoecological studies with high chronological precision are essential to perform detailed comparisons with precisely dated independent evidence such as archaeological findings, historical events, or palaeoclimatic data. Using a new highly-resolved and chronologically precise sedimentary record from Lago di Mezzano (central Italy), we reconstruct decadal-scale vegetation, species diversity, and fire dynamics, aiming to better understand the linkages between climate, land use, fire, and plant communities from the Neolithic to the Copper Age (c. 5100–3100 cal. BC). Closed, mixed beech-oak forests, including evergreen Quercus ilex, dominated the landscape around Lago di Mezzano during the Neolithic and were disturbed by repeated opening phases, with important implications for lake biogeochemistry and mixing regimes. This was in conjunction with increasing fire activity to promote agro-pastoral practices, as inferred from increasing charcoal, Cerealia type, Triticum type, Hordeum type, Plantago lanceolata type, and Urtica pollen. Fires, on their turn, augmented species diversity (richness and evenness). The comparison of the Mediterranean record from Lago di Mezzano with available continuous and high-precision submediterranean and cool-temperate palynological sequences suggests comparable land use pulses across Southern and Central European regions, most likely in connection with climate change. The outcomes of this study are not only of palaeoecological and archaeological interest; they may also help to improve projections of ecosystem dynamics under future global change.