Microalgae biomass has a great potential in search for new alternative energy sources. They can be used as a substrate for the biogas production in anaerobic digestion. When using microalgae, the efficiency of this process is hampered due to the resistant cell wall. In order to accelerate the hydrolysis of cell wall and increase the efficiency of biogas production we applied two different pretreatments - biological and thermal under mesophilic and thermophilic conditions. During biological pretreatment we incubated microalgae with anaerobic hydrolytic bacteria Pseudobutyrivibrio xylanivorans Mz5T. In thermal pretreatment we incubated microalgae at 90 °C. We also tested a combined thermal and biological pretreatment in which we incubated P. xylanivorans Mz5T with thermally pretreated microalgae. Thermal pretreatment in mesophilic and thermophilic process has increased methane production by 21% and 6%, respectively. Biological pretreatment of microalgae has increased methane production by 13%, but only under thermophilic conditions (pretreatment under mesophilic conditions showed no effect on methane production). Thermal-biological pretreatment increased methane production by 12% under thermophilic conditions and by 6% under mesophilic conditions.