Lycoperdon perlatum Pers. (Lycoperdaceae, Agaricales, Agaricomycetidae, Agaricomycetes, Basidiomycota, Fungi) was evaluated for its esterolytic potential. Native electrophoresis of the crude extracts showed four bands having Rf values of 0.34, 0.39, 0.52 and 0.59. The esterase showed the highest activity toward a short-chain substrate, p-nitrophenyl acetate. Optimum reaction conditions for L. perlatum crude extract were attained at pH 8.0 and 40C. Esterolytic activity of enzyme extract was stimulated in the presence of Mn2+, Fe2+, Ca2+ and Zn2+ in the reaction mixture. The enzyme activity was stimulated by incubation at pH 6.0 but retained 77% of its original activity at its optimum pH after 24 h. Thermal inactivation was displayed after incubation for 20 min at various temperatures above 30C. At 1 mM final concentration, 2-mercaptoethanol, dithiothreitol, ethylenediamine tetraacetic acid and p-methylphenyl sulfonylfluoride inhibited the esterolytic reaction. These results support that the crude L. perlatum extract possesses an esterolytic activity having properties similar to other esterases.