Commercially available cellulases and amylases can disperse the pathogenic bacteria embedded in biofilms. This suggests that polysaccharide-degrading enzymes would be useful as antibacterial therapies to aid the treatment of biofilm-associated bacteria, e.g., in chronic wounds. Using a published enzyme library, we explored the capacity of 76 diverse recombinant glycoside hydrolases to disperse Staphylococcus aureus biofilms. Four of the 76 recombinant glycoside hydrolases digested purified cellulose, amylose, or pectin. However, these enzymes did not disperse biofilms, indicating that anti-biofilm activity is not general to all glycoside hydrolases and that biofilm activity cannot be predicted from the activity on pure substrates. Only one of the 76 recombinant enzymes was detectably active in biofilm dispersion, an α-xylosidase from Aspergillus nidulans. An α-xylosidase cloned subsequently from Aspergillus thermomutatus likewise demonstrated antibiofilm activity, suggesting that α-xylosidases, in general, can disperse Staphylococcus biofilms. Surprisingly, neither of the two β-xylosidases in the library degraded biofilms. Commercial preparations of amylase and cellulase that are known to be effective in the dispersion of Staphylococcus biofilms were also analyzed. The commercial cellulase contained contaminating proteins with multiple enzymes exhibiting biofilm-dispersing activity. Successfully prospecting for additional antibiofilm enzymes may thus require large libraries and may benefit from purified enzymes. The complexity of biofilms and the diversity of glycoside hydrolases continue to make it difficult to predict or understand the enzymes that could have future therapeutic applications.