| Summary: | This study investigated the impacts of different brown seaweed species—<i>Ascophyllum nodosum</i>, <i>Sargassum fulvellum, Ecklonia maxima</i>, <i>Lessonia flavicans</i>, <i>Lessonia nigrescens</i>, and <i>Laminaria japonica</i>—on rumen fermentation and methane (CH<sub>4</sub>) mitigation. The current in vitro batch culture study for 24 h at 39 °C evaluated these species in two experimental designs: as feed additive and as feed. The control group for both experimental designs was composed of 500 mg of basal diet (50% grass hay/50% concentrate). For the feed additives experimental design, each seaweed species was evaluated when it was added at 20% of the basal diet, while as a feed, the inclusion level of each species was 20% to partially replace the concentrate in the basal diet as follows (50% hay/30% concentrate/20% seaweed). Chemical analyses showed that the seaweeds were characterized by a high fiber content and high amounts of minerals such as calcium, potassium, and phosphorus, while the protein content ranged within 7 and 13%. When they were applied as feed additives, they increased the production of volatile fatty acids, with <i>L</i>. <i>japonica</i> being the most effective; however, they failed to suppress CH<sub>4</sub> production. In contrast, their inclusion as a feed in the basal diet led to a significant reduction (<i>p</i> < 0.05) in CH<sub>4</sub>, especially for <i>E</i>. <i>maxima</i> and <i>L</i>. <i>japonica</i>, by up to 18 and 21%, respectively, but this was associated with general inhibition of the rumen fermentation. Therefore, the tested seaweeds could be used as a source of minerals and as a feed additive to improve rumen fermentation, but without anti-methanogenic potential. Meanwhile, their inclusion as feed at 20% could reduce CH<sub>4</sub> production with an adverse effect on fermentation. Thus, further trials are needed to identify the appropriate inclusion level to achieve effective CH<sub>4</sub> reduction without any detrimental effects on rumen fermentation.
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