This study was conducted to evaluate the effects of E. stolonifera extract addition on in vitro ruminal fermentation characteristics, methanogenesis and microbial populations. Cannulated Holstein cows (450 ± 30 kg) consuming timothy hay and a commercial concentrate (60:40, w/w) twice daily (09:00 and 17:00) at 2% of body weight with free access to water and mineral block were used as rumen fluid donors. In vitro fermentation experiment, with timothy hay as substrate, was conducted for up to 72 h, with E. stolonifera extract added to achieve final concentration 1, 3 and 5% on timothy hay basis. Administration of E. stolonifera extract to a ruminant fluid-artificial saliva mixture in vitro increased the total gas production. Unexpectedly, E. stolonifera extracts appeared to increase both methane emissions and hydrogen production, which contrasts with previous observations with brown algae extracts used under in vitro fermentation conditions. Interestingly, real-time polymerase chain reaction indicated that as compared with the untreated control the ciliate-associated methanogen and Fibrobacter succinogenes populations decreased, whereas the Ruminococcus flavefaciens population increased as a result of E. stolonifera extract supplementation. E. stolonifera showed no detrimental effect on rumen fermentation characteristics and microbial population. Through these results E. stolonifera has potential as a viable feed supplement to ruminants.