Malted barley waste produced during the brewing process was used as a medium for the growth of Lentinus edodes mycelium. This by-product proved to be a suitable material for mycelial growth and for the production of high levels of laccase and manganese peroxidase, especially when mixed with oak wood chips. Enzyme production by the mycelium grown on a mixture of malt-containing by-product, oak wood chips and perlite was compared in three fermentation systems. High amounts of enzymes were produced in the solid phase in a model solid-state fermentation system, and also in the liquid phase in a system in which the liquid phase was situated under a malt substrate overgrown with L. edodes mycelium in contact with the air.