Ozonation of sewage sludge prior to single-stage anaerobic digestion (SAD) has been reported to increase biogas and/or methane production. However, no studies have evaluated the impact of ozone pre-treatment on two-stage anaerobic digestion (TAD) of sewage sludge over the production of these renewable fuels. Present study, therefore, aimed at investigating the impact of 0.06 g O3/g TSS ozone-pretreated sewage sludge on the productivity of mesophilic semi-batch lab-scale TAD and on its methanogenic consortium. For this purpose, a TAD configuration was constructed by two reactors connected in series to perform acidogenic and methanogenic phases separately. Measurements of methane, biogas and quantification of microbial consortiums were done by gas chromatography, water displacement and fluorescence in situ hybridization techniques, respectively. The ozone pretreatment results highlighted average daily solid removals between 51 and 64%. Ozonation of sewage sludge prior to TAD also yielded 69 and 38% more methane and biogas, respectively. This increase accounted for the predominance shift from strictly hydrogenotrophic to acetotrophic methanogens in the ozonated TAD.