Sargassum spp is an invasive macroalgae and an alternative feedstock for bioethanol production. Sargassum spp biomass was subjected to high-pressure technology for biomass fractionation under different operating conditions of temperature and residence time to obtain glucan enriched pretreated solids (32.22 g/100 g of raw material). Enzyme hydrolysis process at high pretreated solid loading (13%, w/v) and enzyme loading of 10 FPU/g of glucan was performed, obtaining 43.01 g/L of glucose corresponding to a conversion yield of 92.12%. Finally, a pre-simultaneous saccharification and fermentation strategy (PSSF) was performed to produce bioethanol. This operational strategy produced 45.66 g/L of glucose in the pre-saccharification stage, and 18.14 g/L of bioethanol was produced with a glucose to bioethanol conversion yield of 76.23%. The development of this process highlights the feasibility of bioethanol production from macroalgal biomass in the biorefinery concept.