A microalgal biomass offers a potential alternative to the maize silage commonly used in biogas technology. In this study, photoautotrophically grown Scenedesmus obliquus was used as biogas substrate. This microalga has a low C/N ratio of 8.5 relative to the optimum 20–30. A significant increase in the ammonium ion content was not observed. The methane content of the biogas generated from Sc. obliquus proved to be higher than that from maize silage, but the specific biogas yield was lower. Semi-continuous steady biogas production lasted for 2 months. Because of the thick cell wall of Sc. obliquus, the biomass-degrading microorganisms require additional time to digest its biomass. The methane concentration in the biogas was also high, in co-digestion (i.e., 52–56%) as in alga-fed anaerobic digestion (i.e., 55–62%). These results may be related to the relative predominance of the order Clostridiales in co-digestion and to the more balanced C/N ratio of the mixed algal–maize biomass. Predominance of the order Methanosarcinales was observed in the domain Archaea, which supported the diversity of metabolic pathways in the process.