This study aimed to quantitatively determine the composition of 25 essential oils (EOs) using gas chromatography–mass spectrometry (GC-MS) to assess their minimum inhibitory concentrations (MICs) against selected rumen microorganisms and to confirm their effects in in vitro tests on volatile fatty acid (VFA) formation. GC-MS analysis identified over 80 compounds across the tested oils. The MICs were determined for Butyrivibrio fibrisolvens, Prevotella albensis, Lactobacillus delbrueckii ssp. lactis, and Streptococcus bovis, revealing diverse sensitivities. The rumen bacteria’s sensitivity to essential oils varied by strain, with some microorganisms inhibited at low concentrations while others required higher doses, highlighting the potential for targeted modulation of the rumen microbiota. Amyris balsamifera and Zingiber officinale demonstrated strong inhibitory effects at low concentrations and simultaneously enhanced VFA production. In contrast, Lavandula officinalis showed inhibitory effects on VFAs. Amyris balsamifera and L. officinalis also exhibited methane reduction. These findings demonstrate that selected essential oils can modulate rumen microbiota and fermentation by either inhibiting or stimulating specific bacterial groups, highlighting their potential as natural modulators to improve rumen function and animal health.