Pleurotus ostreatus is a widely cultivated and investigated mushroom for its economical and ecological values and medicinal properties. P. ostreatus can be cultivated on different lignocellulosic substrates (oak sawdust, wheat straw, corn cobs and many more). Optimal growth is influenced not only by the composition of the substrate but also by the amount of water in it. In our study, P. ostreatus was cultivated on wheat straw pellets with different water contents (60%, 65%, 70% and 75%). Mycelium growth, biological efficiency (BE), moisture of substrate, pH, enzymatic activities and relationships were the parameters that were evaluated. Based on the results, the optimum initial substrate water content for mycelial growth and BE of the substrate ranged between 65% and 75%. On the other hand, the highest enzymatic activities of hydrolytic and ligninolytic enzymes (Mn-dependent peroxidase, 1,4-β-glucosidase and cellobiohydrolase) were determined for substrates with 75% of water content.