Although the effect of saponins or saponin-containing plants on rumen microorganisms and rumen fermentation has been intensively investigated, this issue still requires special attention. Many of the phenomena occurring in the rumen related to dietary saponin supplementation are still not fully understood. Saponaria officinalis is a triterpenoid saponin-containing plant; thus, the aim of the present study was to evaluate the effect of S. officinalis L. powdered root, methanolic extract of the S. officinalis root (SOR) and the effect of the separated fractions (polysaccharides, saponins and phenolics) of S. officinalis on rumen methanogenesis, microbial population and rumen fermentation characteristics in an in vitro batch culture fermentation system. The powdered root (raw plant material) and S. officinalis extract (SOE) decreased in vitro methane production and consequently reduced the microbial population in a dose-dependent manner. The inhibition of methanogenesis was accompanied by changes in the volatile fatty acids profile. In vitro dry matter digestibility was not affected by any of the secondary compounds applied. The highest applied doses of SOE caused a higher reduction in methanogenesis (33·5 v. 14·4%) than the highest doses of powdered root form. Such results suggest that the basic components of the SOR could interact with phytochemicals or that the phytochemicals became physically less available for microbiota, resulting in a decreased antimethanogenic activity of the powdered root v. the extract. Among all the fractions selected, the saponin fraction exerted the greatest impact on ruminal fermentation. In conclusion, saponins decreased methane production by 29% in comparison with the control. This decrease was related to the reduction in protozoa and methanogen counts. It is proposed that S. officinalis has the potential to inhibit rumen methanogenesis without affecting rumen fermentation adversely.