Enzymatic hydrolysis of olive mill wastewater compounds (OMW) using culture broth of Aspergillus niger, Trichoderma atroviride and Trametes trogii was tested in order to release free simple phenolic compounds having high antioxidant activity. HPLC analysis of treated and untreated OMW indicated the release of large amounts of free hydroxytyrosol (HT). Enzymatic pre-treatment of OMW by A. niger and T. atroviride broth cultures which are rich in β-glucosidase increased the amount of HT respectively from 0.05 up to 1.1 and 0.50g/L. However, the use of T. trogii broth culture showed an oxidation of phenolic compounds due to its high laccase activity. The ethyl acetate extract of OMW treated by A. niger broth culture with IC50 of 45μg/mL showed the strongest antioxidant potency compared to untreated OMW with IC50 of 501μg/mL.