This study aimed to evaluate the effects of increasing doses (0, 10, 20, 40, 80 and 120 mu l/50ml) of essential oil of Eucalyptus (Eucalyptus camaldulensis) on in vitro rumen fermentation. Two 24h-incubations were done and gas production (GP) was measured. The liquid was collected for analysis of ammonia nitrogen (NH3-N) and true organic matter degradability (TOMD). The partitioning factor (PF) was estimated after 24h of incubation. Procedure of H-NMR spectroscopy was used to analyse rumen fluid samples. Results showed that, GP was only affected and decreased significantly for the doses 20, 40, 80 and 120 mu l. Ammonia-N concentration was reduced by 8.2%, 18.6%, 21.6% and 25% when 20, 40, 80 and 120 mu l of EuEO were added respectively. The TOMD values were decreased significantly at doses 40, 80 and 120 mu l. The PF values were affected between doses control and 120 mu l. Concerning H-NMR spectroscopy, results showed that the diet containing 80 mu l of EuEO, there were increases observed in the concentration of rumen methylamine, glucose, butyrate, propionate and glycine. EuEO modified fermentation trends in the rumen, mainly by reducing GP and protein deamination. And the use of EuEO in high concentrations inhibits ruminal flora and decreases the production of VFA.