The aims of this article are, first, to investigate the middle- and upper-Holocene woodland history along the altitudinal gradient between the lowlands and uplands of Central Europe (190–550 m a.s.l.) and, second, to outline possible biases inherent in the charcoal record based on a comparison with the pollen record and its known biases. Our anthracological data set contains 42,547 determinations made in 120 charcoal samples taken at 69 sites. The lowest elevated part of the study region (below 200 m a.s.l.) is characterized by the long-term presence of a species-rich hardwood forest (mixed oak–elm–ash forest). Quercus charcoals dominated in the rest of the altitude zones during the Neolithic and Aeneolithic; however, shrub charcoals appearing in samples from areas with chernozem soils (generally up to 230 m a.s.l.) indicate open-canopy oak woodlands. The species composition differed along the altitudinal gradient during the Bronze Age period, when Carpinus, Fagus and Abies expanded to altitudes above 230 m a.s.l., while Fagus was more abundant above 290 m a.s.l. Broadleaved trees ( Quercus, Fraxinus, Ulmus, Acer and Carpinus ) and shrubs are generally more represented in charcoals than pollen. Since broadleaved trees are usually nutrient demanding and able to re-grow easily after being felled, we suppose that their charcoal record is influenced by two main factors: bias of the initial location of the archaeological site and bias caused by long-term human influence on forest vegetation in the vicinity of settlements. These results underline that combining charcoal and pollen analysis has great potential for studying phenomena in cultural landscapes, as each of the methods approaches nature from the opposite side of the human–nature gradient. Middle- and upper-Holocene woodland history in central Moravia (Czech Republic) reveals biases of pollen and anthracological analysis.