The effects of six essential oils (EO) on rumen fermentation and biohydrogenation were evaluated under in vitro conditions. Three doses (125, 250, and 500 mg/l) of EO were evaluated using in vitro 24 h batch culture of rumen fluid with a 55 : 45 forage : concentrate diet. Treatments were control (CON), control with Siberian fir needle oil (FNO), citronella oil (CTO), rosemary oil (RMO), sage oil (SAO), white thyme oil (WTO), and clove oil (CLO). Treatments were incubated in triplicate in 125 ml flasks containing 500 mg of finely ground total mixed ration (TMR), 25 mg of soybean oil, 10 ml of the strained ruminal fluid, 40 ml of media, and 2 ml of reducing solution. After 24 h, the pH was determined and samples were collected to analyze ammonia N, volatile fatty acids (VFA), and fatty acids (FA). Cultures pH was not affected by EO averaging 6.6 +/- 0.2. In general, high EO doses reduced the total VFA concentration except for SAO and RMO. Relative to CON, all EO decreased (P < 0.05) ammonia N concentrations except for the highest dose of WTO. Except for SAO, EO did not modify acetate to propionate ratio. Relative to CON, the addition of CTO and FNO increased (P < 0.05) the proportions of isobutyrate and decreased (P < 0.05) the proportions of valerate and isovalerate. The concentrations (mg/culture) of C18:0 and C18: 1 trans FA decreased (P < 0.05) with CTO, FNO, RMO, and SAO relative to CON. Most tested EO in this study had little to no effects on conjugated linoleic acids (CLA), and linoleic and linolenic acids concentrations. In conclusion, results from this study showed that except for effects on ammonia N, EO tested in this study had moderate effects on rumen fermentation. The reduction in the formation of trans FA and C18: 0 with some EO may indicate shifts in the biohydrogenation pathways toward the formation of other unidentified intermediate FA.