Estimating past population dynamics has become a major research topic for archaeology, which uses several proxies for studying past demography. The Czech Republic represents a unique region with abundant digital archaeological and palynological datasets comprising tens of thousands of records covering the whole Holocene. We used these datasets to quantify long-term demographic trends and to delimit past human activity in space and time. Our results, based on large data from both disciplines, are one of the first quantitative results covering the whole Czech Republic. Through summed probability distributions of radiocarbon dates and spatio-temporal modelling of human activity based on archaeological sites, we identified four major demographic events and processes between 10,000 BCE and 1000 CE – the beginning of the Neolithic at 5400 BCE, the Eneolithic/Bronze Age transition in the 3rd millennium BCE, Bronze Age expansion after 1500 BCE, and mediaeval expansion after 500 CE. We identified two settlement cores in the lowlands, with additional inner and outer peripheries with different demographic histories. Our study clearly shows that prehistoric and early historic population dynamics were not a homogenised process and were regionalized according to local environmental and social conditions. The comparison of archaeological results with pollen-based vegetation proxies also showed regional aspects in human-vegetation interactions. Agro-pastoral communities dwelling in the lowlands mostly influenced the openness of the landscape, used for fields and pastures, whereas contemporary communities with a similar economic mode residing in a different region are more visible in pollen records through species and structural changes in woodlands. The agro-pastoral subsistence strategy did not start everywhere with the onset of the Neolithic: in some regions it failed after several centuries, in others, the foraging lifestyle persisted significantly longer, and farming became a major economic strategy much later. Our study shows that archaeological site-based and 14C-based demographic proxies cannot be utilized for all periods and regions due to several limitations. Only the combination of different quantitative and qualitative archaeological proxies for population does reveal important details.