Livestock animals are seriously contributing to global warming as methane producers. Six levels (0, 2, 4, 6, 8, and 10 ml.kg^-1 DM) of eucalyptus oil (EuO) were investigated under in vitro conditions to study the mitigation ability for methane production, using two rations: R1 (70% forage: 30% concentrates) and R2 (60% forage: 40% concentrates). Two cannulated sheep were used as donor animals to obtain the rumen liquid. The results showed that CH₄ production levels were significantly (P≤0.05) lower in all treated groups with EuO than the control group (0 ml.kg^-1 DM) in both rations. The retreating for CH₄ of R1 was 32%, adding of 2 mL.kg^-1 DM, and was 38% in10 ml.kg^-1 DM. Regarding R2, the decrease ratio of methane production was 42% in 2.0 mL addition, whereas it was 46% in 10 mL of addition rate. In R2, protozoa count was significantly (P≤0.05) lower by adding the eucalyptus oil compared with the control. In conclusion, using EuO and a high-protein diet could decrease both total gas volume and methane production even with minimal oil levels (2.0 mL EuO.kg^-1DM). It is recommended to carry out an in vivo experiment to emphasize the effects of EuO on the ruminant.