Crude enzyme extracts typically contain a broad spectrum of enzyme activities, most of which are redundant to those naturally produced by the rumen microbiome. Identification of enzyme activities that are synergistic to those produced by the rumen microbiome could enable formulation of enzyme cocktails that improve fiber digestion in ruminants. Compared to untreated barley straw, Viscozyme (R) increased gas production, dry matter digestion (P < 0.01) and volatile fatty acid production (P < 0.001) in ruminal batch cultures. Fractionation of Viscozyme (R) by Blue Native PAGE and analyses using a microassay and mass-spectrometry revealed a GH74 endoglucanase, GH71 alpha-1,3-glucanase, GH5 mannanase, GH7 cellobiohydrolase, GH28 pectinase, and esterases from Viscozyme (R) contributed to enhanced saccharification of barley straw by rumen mix enzymes. Grouping of these identified activities with their carbohydrate active enzymes (CAZy) counterparts enabled selection of similar CAZymes for downstream production and screening. Mining of these specific activities from other biological systems could lead to high value enzyme formulations for ruminants.