The effect of various carbon and nitrogen sources on laccase, manganese-dependent peroxidase (MnP), and peroxidase production by two strains of Pleurotus ostreatus was investigated. The maximal laccase yield of P. ostreatus 98 and P. ostreatus 108 varied depending upon the carbon source from 5 to 62 U l−1 and from 55 to 390 U l−1, respectively. The highest MnP and peroxidase activities were revealed in medium supplemented by xylan. Laccase, MnP, and peroxidase activities of mushrooms decreased with supplementation of defined medium by inorganic nitrogen sources. Peptone followed by casein hydrolysate appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, their positive effects on enzyme accumulation were due to a higher biomass production. The secretion of MnP and peroxidase by P. ostreatus 108 was stimulated with supplementation of casein hydrolysate to the control medium since the specific MnP and peroxidase activities increased 15-fold and 3.5-fold, respectively.