Xylooligosaccharides (XOS) are prebiotic nutraceuticals that can be sourced from lignocellulosic biomass, such as agro-residues. This study reports for the first time an optimization study of XOS production from agro-residues by direct fermentation using two Trichoderma species. A total of 13 residues were evaluated as potential substrates for single-step production. The best results were found for Trichoderma reesei using brewers' spent grain (BSG) as substrate. Under optimal conditions (3 days, pH 7.0, 30 °C and 20 g/L of BSG), a production yield of 38.3 ± 1.8 mg/g (xylose equivalents/g of BSG) was achieved. The obtained oligosaccharides were identified as arabino-xylooligosacharides (AXOS) with degree of polymerization from 2 to 5. One-step fermentation proved to be a promising strategy for AXOS production from BSG, presenting a performance comparable with the use of commercial enzymes. This study provides new insights towards the bioprocess integration, enabling further developments of low-cost bioprocesses for the production of these valuable compounds.