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Fossil fuel depletion has led to an increasing number of research studies and applications focusing on renewable energy, such as different types of biomass. Lignocellulosic biomass represents an abundant source of biomass suitable for energy production in various forms. The present research investigates the application possibility of pedunculate oak bark (Quercus petrea (Matt.) Liebl.) for the production of biogas via anaerobic digestion. This research has significant novelty, as only a few examples on the utilization of tree bark wastes for the production of biogas can be found in the scientific literature. One of the key factors of increasing biogas yield is the efficient hydrolysis of the basic material, which is achieved by different pretreatment methods. In this study, oak bark was pretreated by microwave energy, by extraction, and by the combination of these two methods. The semi-continuous thermophylic anaerobic digestion of untreated oak bark resulted a 76.3 ml/g volatile solid specific methane yield over a 50-day period, which was not significantly lower than methane yield gained from pretreated basic material. Results indicated that oak bark is suitable for the production of biogas even without the application of the investigated pretreatment techniques. As extraction of oak bark does not impair biogas production, the complex biorefinery utilization of oak bark in the form of extraction bark polyphenols and the subsequent anaerobic fermentation of lignocellulosic residue can be accomplished in the future.