A biological pretreatment of olive tree biomass (OTB) was carried out. First, seven white-rot fungi (WRF) were screened on solid-state fermentations by analyzing the substrate composition, ligninolytic enzymes production and enzymatic hydrolysis yields at three different pretreatment times (15, 30 and 45 days). Glucose released by enzymatic hydrolysis of OTB pretreated with Irpex lacteus for 45 days doubled that obtained with the control (non-inoculated). In addition, to enhance this yield, the combination of fungal pretreatment with a chemical pretreatment was studied. It was also found that the order of the pretreatment combination has a relevant effect on the glucose yield. Thereby, the best option determined, fungal pretreatment with I. lacteus after 28 days of culture followed by diluted acid pretreatment (2% w/v H2SO4, 130 °C and 90 min), enhanced 34% the enzymatic hydrolysis yield compared with the acid pretreatment alone. Applying the best pretreatment combination, the overall sugar yield of the whole process (sequential pretreatment plus enzymatic hydrolysis) was 51% of the theoretical yield.