This research was carried out to evaluate the effects of flavone, myricetin, naringin, catechin, rutin, quercetin, and kaempferol at the concentration of 4.5% of the substrate (dry matter basis) on the rumen microbial activity in vitro. Mixture of guinea grass and concentrate (60 : 40) was used as the substrate. The results showed that all the flavonoids except naringin and quercetin significantly () decreased the dry matter degradability. The gas production significantly () decreased by flavone, myricetin, and kaempferol, whereas naringin, rutin, and quercetin significantly () increased the gas production. The flavonoids suppressed methane production significantly (). The total VFA concentration significantly () decreased in the presence of flavone, myricetin, and kaempferol. All flavonoids except naringin and quercetin significantly () reduced the carboxymethyl cellulase, filter paperase, xylanase, and β-glucosidase activities, purine content, and the efficiency of microbial protein synthesis. Flavone, myricetin, catechin, rutin, and kaempferol significantly () reduced the population of rumen microbes. Total populations of protozoa and methanogens were significantly () suppressed by naringin and quercetin. The results of this research demonstrated that naringin and quercetin at the concentration of 4.5% of the substrate (dry matter basis) were potential metabolites to suppress methane production without any negative effects on rumen microbial fermentation.