Integrated bioprocess strategies may facilitate ethanol production from both C6 and C5 fractions of lignocellulosic feedstocks. We propose a new process concept, SSICF, where sugarcane bagasse is hydrolyzed simultaneously with xylose isomerization and the co-fermentation of C6 and C5 sugars. A commercial cocktail was supplemented with our multi-enzymatic system composed of three recombinant enzymes immobilized in Feroxyhyte magnetic nanoparticles: β-glucosidase, β-xylosidase and xylose isomerase. SSICF was performed using non-GMO Saccharomyces at pH 6.0 and 35 °C for 72 h in a synthetic medium containing cellobiose and xylose, and another medium containing pretreated sugarcane bagasse (PSB). The results of ethanol global yields in SSICF were 77.67% and 73.24% for the synthetic medium and PSB, respectively. In a nutshell, this is the first report of a successful proof-of-concept of SSICF with four rounds of enzyme recycling and a non-GMO yeast, an innovative process with high potential for industrial use. Display omitted •A new process concept was proposed for an efficient ethanol production in biorefineries.•The supplementation of immobilized enzymes in the SSICF process was investigated.•Non-GMO Saccharomyces was employed for second-generation ethanol production from PSB in SSICF.•The multi-enzymatic system allowed the yeast to produce ethanol from both C5/C6 sugars.•Ethanol production was achieved in all SSICF cycles with enzyme recycling.