The biological pretreatment of lignocellulosic biomass for the production of bioethanol is an environmentally friendly alternative to the most frequently used process, steam explosion (SE). However, this pretreatment can still not be industrially implemented due to long incubation times. The main objective of this work was to test the viability of and optimise the biological pretreatment of lignocellulosic biomass, which uses ligninolytic fungi (Pleurotus eryngii and Irpex lacteus) in a solid-state fermentation of sterilised wheat straw complemented with a mild alkali treatment. In this study, the most important parameters of the mechanical and thermal substrate conditioning processes and the most important parameters of the fungal fermentation process were optimised to improve sugar recovery. The largest digestibilities were achieved with fermentation with I. lacteus under optimised conditions, under which cellulose and hemicellulose digestibility increased after 21 days of pretreatment from 16 to 100 % and 12 to 87 %, respectively. The maximum glucose yield (84 %) of cellulose available in raw material was obtained after only 14 days of pretreatment with an overall ethanol yield of 74 % of the theoretical value, which is similar to that reached with SE. © 2012 Springer-Verlag Berlin Heidelberg.