Effects of wormwood essential oil (EO) supplementations on in vitro digestibility and rumen fermentation characteristics of soybean meal (SBM) or bermudagrass hay (BH) were examined. Rumen fluid was collected from 2 cannulated Holstein cows, filtered through 2 layers of cheese cloth and mixed with Van Soest medium (1:2 ratio). Exactly 0.5 g of SBM or BH was treated with nothing or with 200 μL of wormwood EO from Ganghwa (GA), Injin (IN) or San (SA) wormwood hybrids grown at the Research Unit, Gyeongsang National University, South Korea. Three replicates of each treatment with 3 blanks were incubated at 39°C for 72 h in buffered-rumen fluid (40 mL). In vitro DM digestibility (DMD), pH, ammonia-N and volatile fatty acids (VFA) were measured. The population of certain rumen microbes was measured by Real-time PCR. Data were analyzed using the GLM procedure of SAS (SAS Inst., Cary, NC). Total VFA concentration (mM) of SBM was decreased (P < 0.05) by GA and SA (157 vs. 143, and 146) but not IN (149). Other fermentation or digestibility measures and the methanogenic archaea population were unaffected by EO. However, the fold change of Fibrobacter succinogens was reduced (P < 0.05) by GA, IN and SA (18.8 vs. 9.13, 2.36, 2.63), that of Ruminococcus albus was increased (P < 0.05) by SA (10.8 vs. 24.5), that of Streptococcus bovis was increased (P < 0.05) by GA and SA though greatest by SA (P < 0.05) and that of R. flavefaciens was increased (P < 0.05) by GA and IN. The DMD of BH was not increased by EO but GA decreased NDF digestibility. Ammonia-N concentration was increased (P < 0.05) by SA (17.7 vs. 19.9 mg N/dl). Total VFA concentration was reduced (P < 0.05) by GA (136.1 vs. 119.8 mM) but pH and VFA molar proportion did not differ among treatments (P > 0.05). Adding the EO to SBM or BH had no beneficial effects on the fermentation or digestibility despite altering the ruminal bacteria population.