Despite recent improvements, second-generation (2G) biorefineries still have challenges to overcome, especially regarding cellulose and xylan hydrolysis and xylose fermentation. Hydrolases account for a high percentage of the process' total cost, and industrial yeasts may not assimilate xylose properly. In this sense, prospecting microorganisms that can both metabolize this sugar and hydrolyze cellulose and xylan may lead to significant advances in 2G processes. Since the gut microbiota of herbivorous insects has proven to be the major player in their lignocellulose digestion, yeasts were isolated from the gut of Spodoptera frugiperda larvae, and their cellulolytic, xylanolytic, and xylose-assimilating capacity were analyzed. From a hundred larvae guts, forty-six yeasts were selected for their growth capacity in xylose-containing media. Fourteen strains were able to hydrolyze cellulose, and three showed xylanolytic activity. Two strains, taxonomically identified as Papiliotrema laurentii and Meyerozyma caribbica, stood out with the highest cellular biomass yield and productivity when submitted to a high-throughput microscale growth analysis under different culture conditions (varying pH and sugar concentration). While the P. laurentii strain displayed the highest xylanolytic activity (205.48 U/mL) and an intermediate cellulolytic capacity (31.25 U/mL), the M. caribbica strain showed, besides cellulase activity (37.46 U/mL), high ethanol and xylitol yields (0.36 g(ethanol)/g(glucose) and 0.35 g(xylitol)/g(xylose), respectively) in media with the most common fermentation inhibitors in 2G bioprocesses. Through a Central Composite Rotatable Design, the optimized condition for the simultaneous production of ethanol and xylitol was found to be in a 55-85 g/L range of sugar concentration and between pH values of 6.0 to 8.5. Also, this experimental design analysis allowed the validation of empiric-coded models. These results indicate the high biotechnological potential of prospecting yeasts in S. frugiperda guts.