Plantain (PL) contains plant secondary metabolites (PSM), such as acteoside, aucubin, and catalpol, known for their bioactive properties. While acteoside and aucubin have been linked to reducing nitrogen losses in grazed pastures, their effects on enteric methane (CH4) emissions remain unexplored. Three in vitro batch culture experiments were conducted to assess the effects of PSM on rumen fermentation, using PL pastures with varying PSM concentrations, purified PSM compounds, and/or their combinations added to ryegrass (Lolium perenne, RG), which does not contain these PSM. Aucubin addition to RG extended the time to reach halftime for gas production (GP) and CH4 by 15-20% due to its antimicrobial effects. Acteoside, alone or with aucubin, promoted propionate production, an alternative hydrogen sink, which reduced the acetate to propionate ratio, increased GP by up to 13%, and decreased CH4 proportion in gas by 5-15%. Aucubin reduced ruminal net ammonia (NH3) production by up to 46%, with a similar reduction observed when combined with acteoside. This study highlights the potential of PSM to mitigate CH4 emissions and reduce nitrogen losses from dairy cows, warranting in vivo evaluation of PSM and targeted breeding of PL pastures with increased PSM content.