Abstract <p>Presence of carbohydrates hampers protein degradation in anaerobic digesters. To understand this phenomenon, we used proteogenomics to identify the active protein-degraders in the presence of low and high carbohydrates concentrations. Active metabolic pathways of the identified protein-degraders were investigated using proteomics with ¹³C-protein substrates (protein stable isotope probing). Results showed that 1) <i>Acinetobacter</i> was the active protein-degraders under both protein-fed and protein-glucose mixture-fed conditions, 2) the relative abundance of <i>Acinetobacter</i> was not affected by the presence of carbohydrates, 3) the incorporation of the ¹³C-labelled protein substrate was predominantly observed in outer membrane-bound proteins and porin proteins, which are associated with proteinases or the transportation of amino acids across the cell wall. The <i>Acinetobacter</i> metabolic model and the incubation conditions suggested that glucose and proteins were degraded through anaerobic respiration. The negative impact of carbohydrates on protein biodegradation was attributed to <i>Acinetobacter</i>'s preference for carbohydrates. This work highlights that efficient degradation of protein and carbohydrate mixtures in anaerobic digesters requires a staged or time-phased approach and enrichment of active protein-degraders, offering a new direction for process optimization in anaerobic digestion systems.</p> Key points <p>• <i>Acinetobacter identified for the first time as main anaerobic protein-degrader</i></p> <p>• <i>Metabolic model revealed protein degradation via anaerobic respiration</i></p> <p>• <i>Metabolic pathway analysis indicated SO</i>_<i>4</i>^<i>2−</i><i> or Fe</i>^<i>3+</i><i> as terminal electron acceptors</i></p>