In the present study, the in-situ produced fungal mash rich in hydrolytic enzymes was used for the pretreatment of activated sludge, food waste and their mixture prior to anaerobic digestion. The enzymatic pretreatment of activated sludge mixed with food waste resulted in the production of 3.72 g/L glucose and 51 mg/L free amino nitrogen, equivalent to 7.65 g/L soluble chemical oxygen demand (SCOD) within 24 h, accompanied with 19.9% of volatile solids (VS) reduction. After pretreatment of activated sludge and food waste by fungal mash, 19.1% and 21.4% of VS reduction were achieved respectively. Furthermore, the bio-methane yield of mixed waste pretreated with fungal mash was found to be 2.5 times higher than activated sludge without pretreatment, with a further VS reduction of 34.5%. These suggest a total VS reduction of 54.3% in the proposed anaerobic system with the pretreatment by fungal mash. Theoretical estimation further revealed that about 678 million kW h of electrical energy could be potentially recovered annually through the co-digestion of mixed waste activated sludge and food waste after the pretreatment with fungal mash in Singapore. In this case, the energy produced was higher than energy consumed by wastewater treatment. It was demonstrated in this study that the pretreatment of mixed activated sludge and food waste by in-situ produced fungal mash would be a promising option for enhancing biomethane production as well as for maximizing volume reduction of mixed waste via anaerobic co-digestion. (C) 2016 Elsevier Ltd. All rights reserved.