In this work, the application of ozone, an unexplored pretreatment to improve methane production from three mixed microalgae sources was investigated. Ozonation at various, culture-dependent dosages was employed and its impact on the efficiency of subsequent anaerobic digestion was examined in comparison with untreated biomass. Regardless of the O-3 pretreatment conditions, the methane yield in all cases could be increased by various extents (6-66%). The highest methane production (432.7 mL CH4 g(-1) VSalgal, equaling to 259.6 mL CH4 g(-1)COD(in)) was attained with the culture composed of 35% Scenedesmus, 30% Dictyosphaerium, 15% Keratococcus, 10% Oscillatoria, 8% Monoraphidium and 2% Nitzchia, using the highest dosage of ozone (382 mg O-3 g(-1) VSalgal) for pretreatment. Electron microscopic assessment revealed the devastating effect of O-3 on algal cell wall structure and integrity, which was thus identified as the key-contributor to the improvement of CH4 fermentation. (C) 2016 Elsevier B.V. All rights reserved.