The formation of hydrogen sulfide (H2S) during anaerobic digestion (AD) imposes constraints on the valorisation of biogas. So far, inorganic sulfur compounds -mainly sulfate - have been considered as the main contributors to H2S formation, while the contribution of organic sulfur compounds is mostly neglected. This study investigates the fate of organic and inorganic sulfur compounds during two-stage anaerobic digestion with intermediate thermal hydrolysis for treatment of primary and secondary sludge in a WWTP treating domestic wastewater. The results of a seven-week monitoring campaign showed an overall decrease of organic sulfur compounds in both stages of anaerobic digestion. Further fractionation of organic sulfur revealed a high conversion of the particulate organic fraction during the first digestion stage and of the soluble organic fraction during the second digestion stage. The decrease of soluble organic sulfur during the second digestion stage was attributed to the solubilisation and hydrolysis of sulfur-containing organic compounds during thermal hydrolysis. In both digestion stages, more organic sulfur was taken up than particulate inorganic:sulfur (metal sulfide) was produced, indicating the formation of other reduced sulfur forms (e.g. H2S). Further batch experiments confirmed the role of organic sulfur uptake in the formation of H2S during anaerobic digestion as sulfate reduction only partly explained the total sulfide formed (H2S in biogas and precipitated FeS). Overall, the conversion of organic sulfur was demonstrated to play a major role in H2S formation (and thus the biogas quality), especially in case of thermal hydrolysis pretreatment.