The aim of this study was to assess the effects of five eucalyptus species (Eucalyptus camaldulensis, E. leucoxylon, E. astringens, E. sideroxylon, and E. lehmannii), harvested in spring, autumn, or winter from two Tunisian arboretums, on in vitro rumen fermentation and methanogenesis. Batch cultures were performed to determine rumen fermentation kinetics and end-product formation after 24 h of incubation. The foliage of the species E. sideroxylon and E. lehmannii showed the greatest digestibility coefficients, whereas E. leucoxylon was the least digestible. Acetate-to-propionate ratio was reduced when E. sideroxylon and E. lehmannii were incubated, and these species were also the most efficient at reducing methane emission. Foliage harvested in winter showed greater digestibility and an increase in the acetate-to-propionate ratio than in other seasons, without an increase in methane emission. Foliage from E. sideroxylon and E. lehmannii showed a potential to decrease enteric methane production without depressing effects on ruminal fermentation. In vivo studies would be necessary to conclusively validate these effects as the first step towards proposing the inclusion of eucalyptus leaves in ruminant diets.