Farm ruminants are responsible for about 15 percent of total global methane emission due to enteric fermentation of feeds. Additives are being used to modify rumen fermentation towards reduced methanogenesis, however, hazards associated with feeding of antibiotics and other chemical compounds to livestock for improving animal performance and methane mitigation are growing public awareness and encourage the research towards exploiting natural products as feed additives. Therefore, an experiment was carried out to examine the potential of eucalyptus (Eucalyptus citriodora) leaves essential oils in modulating rumen fermentation for reduced methane production in water buffalo (Bubalus bubalis). Essential oils were extracted both from dried and fresh eucalyptus leaves, separately in Soxhlet's apparatus by organic solvent, hexane [15 g leaves extracted in 100 ml n-hexane (mol. wt. 86.18, assay 99.0 percent and density 0.658)] for 72 h and tested at three dose levels (0, 0.5 and 2.0 ml) in buffered rumen fluid (BRF). Oats hay (200+-5 mg) was used as substrate and incubated with 30 ml BRF in 100 ml calibrated glass syringes at 39 deg C for 24 h following standard in vitro gas production protocol. Gas production was recorded by displacement of piston in the syringe. Methane in the gas phase and volatile fatty acids in the fermentation medium were estimated by Gas Chromatograph (GC). The total gas production and dry matter digestibility was increased (P LT 0.05) at low dose (0.5 ml) of eucalyptus leaves extract, irrespective of nature of leaves however, these were reduced (P LT 0.05) at higher dose (2.0 ml). Methane production was reduced (P LT 0.05), irrespective of doses. Volatile fatty acids (VFA) production were increased (P LT 0.05) at low dose but higher dose detrimentally reduced it. Rumen ammonia - N was not affected at low dose however, it was reduced at high dose of extract. Extracts from both dried and fresh leaves behaved similarly in modulating all the rumen fermentation parameters at corresponding dose levels. Results of this study revealed reduced methane production with increased feed digestibility and volatile fatty acids production by dosing extract of eucalyptus leaves. Therefore, it suggests using of either extract of eucalyptus (Eucalyptus citriodora) leaves (0.5 ml/30 ml rumen fluid) or its corresponding dose of leaves as feed additive in buffaloes.