The influence of ultrasonication pretreatment on fermentative bioenergy [ethanol/hydrogen (H2)] production from a newly isolated microalgae biomass (Scenedesmus obliquus YSW15) was investigated. S. obliquus YSW15 biomass was sonicated for 0 min (control), 5 min (short-term treatment), 15 and 60 min (long-term treatment), which caused different states of cell lysis for microbial fermentation. Long-term sonication significantly damaged the microalgal cell integrity, which subsequently enhanced the bioenergy production. The accumulative bioenergy (ethanol/hydrogen) production after long-term sonication was almost 7 times higher than that after short-term treatment or the control. The optimal ratio of microalgal biomass to anaerobic inoculum for higher bioenergy production was 1:1. Microscopic analyses with an energy-filtering transmission electron microscope (EF-TEM) and an atomic force microscope (AFM) collectively indicated that cells were significantly damaged during sonication and that the carbohydrates diffused out of the microalgae interiors and accumulated on the microalgae surfaces and/or within the periplasm, which led to enhanced bioaccessibility and bioavailability of the biomass. These results demonstrate that ultrasonication is an effective pretreatment method for enhancing the fermentative bioenergy production from microalgal biomass.