The economic management of lignocellulosic biomass from semi-natural grasslands is now a challenge across Europe. The abandonment of mowing these grasslands leads to the gradual degradation of these ecosystems. This study investigates how chemical and biological factors affect the suitability of biomass from abandoned grasslands for biogas production. We sampled 30 mown and 30 abandoned grassland sites in the Sudetes Mountains (Poland and Czechia). The cover contribution of short herbs was found to be significantly higher in mown grasslands (p < 0.001), while that of tall herbs was more prevalent in abandoned grasslands (p < 0.01). The specific biogas yield (SBY, NL kg−1 volatile solids) is negatively affected by an increased percentage of herbs in the biomass of mown and abandoned grasslands. This is due to the inhibitory effect of herbs on biodegradation, the increase in lignin content and the decrease in cellulose. This study highlights the importance of individual plant species in assessing grassland biomass for area biogas yield (ABY, m3 ha−1) and provides new insights into a field that has not yet been extensively investigated. In mown grasslands, ABY was most positively correlated with grass species (Arrhenatherum elatius, Trisetum flavescens and Festuca pratensis). In abandoned grasslands, the ABY was most correlated with herbaceous species (Galium aparine, Urtica dioica and Chaerophyllum aromaticum) and grasses (A. elatius and Elymus repens). Mown grasslands had significantly higher species richness (p < 0.001) compared to abandoned grasslands, but the number of species sampled did not correlate with SBY and ABY. This study contributes to the development of a sustainable bio-economy by highlighting the need for efficient use of grassland biomass. This approach helps protect semi-natural ecosystems and facilitates sustainable management of renewable resources.