Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular spaces of plant tissues. Fungal hyphae are surrounded by an inner, insoluble cell wall layer and an outer, soluble extracellular polysaccharide (EPS) matrix. Here we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, implicating different functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic GH17 family, is not active on fungal walls, but releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose substituents and can scavenge reactive oxygen species via oxidative self-degradation. Additionally, exogenous application of β-GD leads to enhanced fungal colonization in barley. Our data highlights the hitherto undescribed capacity of this often-overseen fungal EPS layer to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.