Thermophilic Neosartorya fischeri P1 is an excellent carbohydrate-active enzyme (CAZyme) producer. Two alpha-galactosidases of GH (glycoside hydrolase) family 27 with a very low sequence identity (28.7 %), Gal27A and Gal27B, were identified in strain P1 and functionally expressed in Pichia pastoris. In comparison to other characterized GH27 fungal counterparts, rGal27B has a higher temperature optimum (75 degrees C) and better thermostability (>50 % activity at 70 degrees C for 15 min), and rGal27A shows stability over the broadest pH range (pH 2.0-12.0). Moreover, great distinctions lie in the two enzymes. When using pNPG as the substrate, rGal27B had a higher turnover number (1621.4 vs. 368.3 s(-1)) but lower affinity (2.84 vs. 0.8 mM) and catalytic efficiency (460.8 vs. 580.3 s(-1) mM(-1)) than rGal27A. rGal27B acted on galacto-oligosaccharides, whereas rGal27A was active on polymeric substrates. Although both enzymes showed synergy in galactomannan degradation when combined with a beta-mannanase of the same strain, enzyme combinations including rGal27A released more reducing sugars (up to 11.67-fold). Homology modeling predicts different loops in N. fischeri alpha-galactosidases, highlighting the larger tunnel structure in Gal27A to accommodate/bind branched galactomannan with high galactose contents. Phylogenetic analysis reveals the far relationship of Gal27A and Gal27B that they may evolve in different action modes, and their coexistence widens the substrate spectrum for nutrient utilization. This study illustrates the substrate profiles and synergistic mechanism of GH27 alpha-galactosidases of different structures.