This study employed two commercial enzyme preparations to examine the effects of endoglucanase, xylanase or their combination on in vitro volatile fatty acid (VFA) production by ruminal microbial populations. Batch ruminal cultures were established with one of various feedstuffs or with a fescue hay-based diet and ruminal fluid from a heifer fed a 40% forage:60% concentrate diet. Addition of xylanase at 135 xylanase units (XU) per ml increased total VFA production from the fescue hay-based diet (44.3 vs. 57.2 mM, p < 0.05) without changing the acetate to propionate (A:P) ratio. Addition of endoglucanase at 2, 3, 4, and 5 carboxymethyl cellulase units (CMCU) per ml increased total VFA production from the fescue hay-based diet on average by 36% (p < 0.05). Addition of 3, 4 and 5 CMCU/ml also decreased (p < 0.05) the A:P ratio. The combined addition of xylanase (135 XU/ml) and endoglucanase (5 CMCU/ml) increased total VFA production from the fescue hay-based diet (40.9 vs. 61.5 mM, p < 0.05) and reduced the A:P ratio (3.4 vs. 1.5, p < 0.05). The effects of endoglucanase and xylanase supplementation on in vitro VFA production varied across the various substrates used. However, endoglucanase supplementation consistently reduced the A:P ratio with all substrates tested. The effects of the enzyme combination were generally greater than either enzyme alone. We conclude that endoglucanase and xylanase activities differ in their ability to affect ruminal VFA production, and endoglucanase but not xylanase, may improve fermentation efficiency by reducing the A:P ratio.