Abstract Background The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides. Results Large-scale screening identified a range of hydrolytic activities among 348 unique isolates representing 156 species of plant pathogenic and non-pathogenic fungi. Hierarchical clustering was used to identify groups of species with similar hydrolytic profiles. Among moderately and highly active species, plant pathogenic species were found to be more active than non-pathogens on six of eight substrates tested, with no significant difference seen on the other two substrates. Among the pathogenic fungi, greater hydrolysis was seen when they were tested on biomass and hemicellulose derived from their host plants (commelinoid monocot or dicot). Although T. reesei has a hydrolytic profile that is highly active on cellulose and pretreated biomass, it was less active than some natural isolates of fungi when tested on xylans and untreated biomass. Conclusions Several highly active isolates of plant pathogenic fungi were identified, particularly when tested on xylans and untreated biomass. There were statistically significant preferences for biomass type reflecting the monocot or dicot host preference of the pathogen tested. These highly active fungi are promising targets for identification and characterization of novel cell wall degrading enzymes for industrial applications.