The administration of Bacilli to dairy cows exerts a beneficial effect on dry matter intake, lactation performances and milk composition. In this work, two isogenic strains, only differing in the se-cretion level of cellulolytic and xylanolytic enzymes, were compared to establish whether B. subtilis cellulase and xylanase enzymes are involved in the positive effect exerted by these bacteria on animal nutrition. Conveniently, bacteria were grown in a newly designed growth medium based on an inexpensive waste. A multi-factorial study was conducted in which eight feed in-gredients were treated in vitro, using ruminal fluid from cannulated cows, with cultures of the two B. subtilis strains. Feed digestibility and gas production, a parameter linked to feed fermentability, were assessed. Fiber degradability was significantly higher in feeds treated with the en-zyme-overexpressing strain (10% and 5% higher than control treatment with B. subtilis-free media and the parent strain, respectively). This effect was accentuated for the most recalcitrant feeds, in which the benefits of cellulolytic and xylanolytic enzymes were maximized. Fermentability also benefited by the treatment with the enzyme-overexpressing strain, although the trend observed in gas production didn’t reach statistical difference between the two strains, and only minor changes in fermentation profiles were detected. Our results revealed that B. subtilis cellulases and xylanases effectively contribute to improving forage quality, supporting the use of Bacilli as fodder sup-plements to increase animal productivity.