Mitigation of the methane (CH4) emission from ruminants is needed to decrease the environmental impact of ruminant animal production. Different plant materials and chemicals have been tested, but few are both effective and practical. Medicinal herbs contain biological compounds and antimicrobials that may be effective in lowering the CH4 production. However, few studies have systematically evaluated medicinal herbs for their effect on CH4 production or on the rumen microbiota. In this study, extracts from 100 medicinal herbs were assessed for their ability to decrease CH4 production by rumen microbiota in vitro. The extracts of 12 herbs effectively lowered the CH4 production, with the extract of Perilla frutescens seeds being the most effective. The major components of P. frutescens seed extract were identified, and the effects of the extract on the fermentation characteristics and populations of rumen methanogens, fungi, protozoa, and select bacteria were also assessed. The decreased CH4 production induced by the P. frutescens seed extract was accompanied by an increased abundance of Ruminobacter, Selenomonas, Succinivibrio, Shuttleworthis, Pseudobutyrivbrio, Anaerovibrio, and Roseomonas and a decreased abundance of Methanobrevibacter millerae. The abundance of Pedobacter, Anaeroplasma, Paludibacter, Ruminococcus, and unclassified Lachnospiraceae was positively correlated with the CH4 production, with no effects on volatile fatty acids. This study suggests that medicinal herbs may be used to mitigate the CH4 emission from ruminants.