Continuous bio-methanization of different feedstocks (rye grain silage, maize silage, feed residue (mix of silages), solid cattle manure, and grass silage) was investigated in a long-term laboratory-scale experiment with and without enzyme application. Ten-liter reactors were operated simultaneously in a two-step digestion mode for the continuous production of biogas from different feedstocks over 354 days. One set of reactors was operated as main digester, while the second set was used for the second step. The daily input of feedstock was increased from an organic loading rate of 1 to 3 kg ODM.m(-3).d(-1). All digesters were run under stable conditions, indicated by the ratio of volatile fatty acids to the total inorganic carbon, ranging around 0.2 in the first step and 0.15 in the second step. The hydraulic retention time was maintained between 80 and 90 days during the experiment. The application of enzymes was able to enhance biogas production by 10-15% and increase the methane content of biogas by an increment of 5-10% for the investigated materials except for feed residue. The increase in biogas yields was also reflected in the change in the ratios of heating values of the methane produced to the dry materials. These ratios ranged between 0.43 and 0.71 for the untreated feedstock, increasing to 0.44-0.88 after enzyme application.