BackgroundGrape and winery by-products have nutritional values for cattle and also contain functional compounds like phenols, which not only bind to protein but can also directly affect microbiota and their function in the rumen. We characterized the nutritional and functional effects of grape seed meal and grape pomace as well as an effective dosage of grape phenols on ruminal microbiota and fermentation characteristics using a rumen simulation technique.ResultsSix diets (eachn= 8) were compared including a control diet (CON, no by-product), a positive control diet (EXT, CON + 3.7% grape seed extract on a dry matter (DM) basis), two diets with grape seed meal at 5% (GS-low) and 10% (GS-high), and two diets with grape pomace: at 10% (GP-low) and 20% (GP-high), on a DM basis. The inclusion of the by-product supplied total phenols at 3.4%, 0.7%, 1.4%, 1.3%, and 2.7% of diet DM for EXT, GS-low, GS-high, GP-low, and GP-high, respectively. Diets were tested in four experimental runs. All treatments decreased ammonia concentrations and the disappearances of DM and OM (P< 0.05) compared to CON. EXT and GP-high lowered butyrate and odd- and branch-chain short-chain fatty acids while increased acetate compared to CON (P< 0.05). Treatments did not affect methane formation. EXT decreased the abundance of many bacterial genera including those belonging to the core microbiota. GP-high and EXT consistently decreasedOlsenellaandAnaerotipeswhile increasedRuminobacterabundances.ConclusionThe data suggest that the inclusion of winery by-products or grape seed extract could be an option for reducing excessive ammonia production. Exposure to grape phenols at a high dosage in an extract form can alter the rumen microbial community. This, however, does not necessarily alter the effect of grape phenols on the microbial community function compared to feeding high levels of winery by-products. This suggests the dominant role of dosage over the form or source of the grape phenols in affecting ruminal microbial activity. In conclusion, supplementing grape phenols at about 3% of diet DM is an effective dosage tolerable to ruminal microbiota.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40104-023-00892-7.