The aim of this study was to evaluate the effect of contaminants in residues from biogas production (RBP) on soil microbiological and biochemical properties. The RBP, originating from three biogas plants with different residue treatments, were used in two types: the unseparated fraction and the separated (solid) fraction. The experiment was conducted over a period of 1 or 825 days in pots, adding RBP at a 1 % rate. The addition of RBP to the soil stimulated the number of culturable bacteria and fungi (from 12 % to 10 times) and enzymatic activity (from 38 % to 44 times). In short-term incubation, only phosphatases did not show a statistically significant (P > 0.05) change in activity. Separation of the RBP into solid and liquid fractions significantly impacted activity compared to the unseparated material. The total number of culturable bacteria and the number of bacteria participating in the nitrogen transformation cycle were lower (from 1.2 to 10 times), while the number of fungi and the enzymatic activity were higher (from 1.5 to 6.5 times) for separated RBP in comparison to unseparated materials. A significant relationship between the number of nitrifying and denitrifying bacteria in the amended soil and the content of PAHs was also observed. As far as the long-term incubation of RBP with the soil is concerned, it was found that PAHs (P ≤ 0.05) and Cr (P ≤ 0.05) present in RBP had an inhibiting impact on activity of dehydrogenases and urease, respectively, whereas light PAHs (P ≤ 0.05) were found to have a positive effect on the activity of alkaline phosphatase as well as on the number of fungi and bacteria.