Semi-natural grassland habitats are most often limited to animal grazing and low intensity farming. Their potential in bioenergy production is complicated due to the heterogeneity, variation, accessibility, and need for complex pre-treatment/hydrolysis techniques to convert into valuable products. In this research, fermentable sugar production efficiency from various habitats at various vegetation periods was evaluated. The highest fermentable sugar yields (above 0.2 g/g volatile solids) over a period of 3 years were observed from habitats “xeric and calcareous grasslands” (Natura 2000 code: 6120) and “semi-natural dry grasslands and scrubland facies on calcareous substrates” (Natura 2000 code: 6210). Both had a higher proportion of dicotyledonous plants. At the same time, the highest productivity (above 0.7 t sugar/ha) was observed from lowland hay meadows in the initial stage of the vegetation. Thus, despite variable yield-affecting factors, grasslands can be a potential resource for energy production.