The emergence and early development of agro-pastoral activities in Mediterranean coastal areas were key drivers of ecosystem change. While the role of anthropogenic pressures is now well understood, the identification and chronology of the first human imprints on ecosystem dynamics is still an open debate in several countries, especially in the northeastern Adriatic where there is a paucity of palaeoecological data. For the period 5200-3200 cal BP, we here report a comprehensive and integrative study of Northern Adriatic Croatia (Istrian Peninsula, Busuja Bay), showing that: (i) significant human impacts on ecosystems began around 5000 years ago, during the Chalcolithic; (ii) anthropogenic pressures (agriculture and human-induced fire) were the main drivers of long-term ecological change; (iii) local coastal shifts (higher seawater inputs in the sheltered bay) also played a decisive role in ecosystem dynamics; and (iv) climate pressures compounded human impacts. We also suggest that wild olive trees (Olea europaea subsp. europaea var. sylvestris) were an integral part of early diversified agriculture, 500 years before the Bronze Age.