A multi-enzymatic biopreparation of Trichoderma atroviride G79/11 origin was characterized. The fungus showed relatively high cellulase production in a soybean flour-cellulose-lactose medium. Subsequently, based on its post-culture liquid, the biopreparation of the enzyme mixture was developed and characterized. The liquid form of the enzyme mixture reached 22 U cm-3 of cellulolytic activity and its lyophilisate exhibited 1.09 U cm-3 at pH 5.1 and 50 °C. The enzyme mixture was characterized by the following activities: xylanase, β-glucosidase, carboxymethyl cellulase, polygalactouronase, pectinesterase, amylase, lactase, and protease. A method for an efficient conditioning process of organic waste (fruit processing waste, dairy sewage sludge, corn silage, and grain broth) for biogas yield enhancement using the enzyme mixture was proposed. The enzyme mixture increased the efficacy of biogas production by 30% when the lyophilizate (0.5 mg g-1 d.m.) was applied prior to fermentation. A method for conducting the enzymatic conditioning process of organic waste using the enzyme mixture as a pretreatment was proposed. This was part of the optimization of the methane fermentation process to increase the biogas yield. Consequently, after application of the biopreparation, the efficiency of anaerobic digestion of organic waste was improved.