(Acetyl)cholinesterase is an enzyme which is mostly regarded as important in the nervous system signaling. It is found in colinergic sinapses of the animals where it breaks down the neurotransmitter acetylcholine. Doing that, it terminates singaling transmission between neurons. In the last decade, many other acetylcholinesterase roles have been discovered; for example roles in cell differentiation and communication and immune response. Considering that cholinesterases and/or acetylcholine have recently been discovered in plants, different microorganisms and mold Aspergillus niger and Physarum polycephalum, it is only fair to expect finding them in higher fungi. At present, there hasn't been any research that tackled this kind of subject. Within this thesis, cholinesterase activity was determined in water extracts of various Basidiomycota using colorimetric test (Ellman's method), denaturating and native electrophoresis. Ellman's method showed the strongest enzyme activity when substrate acetylthiocholine chloride was used as opposed to butyrylthiocholine chloride and propionyltiocholine chloride. Polyacrylamide gel electrophoresis containing sodium dodecyl sulphate (SDS-PAGE) hasn't delivered wanted results, however native electrophoresis confirmed the results obtained using Ellman's method in which samples G18 (Cortinarius variicolor) and G32 (Echinoderma echinaceum) show the highest activity. These kind of results may give new directions in experimenting with new kinds and roles of cholinesterases.