β-glucan is a polysaccharide that provides beneficial properties to the consumers. Hydrolysis of β-glucans by specific β-glucanases can generate gluco-oligosaccharides which maintains or even increase its original biological activity. In this study, β-(1→3)-glucanases from Trichoderma reesei QM 9414 and Trichoderma harzianum Rifai were applied on the hydrolysis of oat flour aiming production of prebiotic sugars. Hydrolysis was carried out using 20 U of crude enzyme/0.5g of oat flour, pH 5.0, 24 h at 35 ºC. The hydrolyzed oat flour was subjected to evaluation of prebiotic activity by microbiological assay using strains of Lactobacillus reuteri and Lactobacillus acidophilus. Approximate 1x107 UFC/mL of pre-inoculum were cultivated in broths of Man, Rogosa, Sharpe (MRS), containing different carbon sources (1 %): oat flour, glucose, hydrolyzed flour by T. reesei QM 9414 and T. harzianum Rifai, separately. Growth was assessed after 24 and 48 h at 37 °C in anaerobic conditions. The hydrolysis of oat flour resulted in 36.64 and 68.75 % of reducing sugar in 24 h, using β-(1→3)-glucanases from T. harzianum Rifai and T. reesei QM 9414, respectively. The evaluation of prebiotic activity, based on microbial growth showed peaks after 24 h growth in all carbon sources. However, the L. acidophilus shown higher growth in medium supplemented with hydrolyzed oat flour compared to others sugars and to L. reuteri. The results confirm the presence of gluco-oligosaccharides with prebiotic activity in the mixture of hydrolyzed oat flour.