In this study, in vitro ruminal fermentation, anti-methanogenesis, and ammonia formation of two autotrophic algae [Nannochloropsis gaditana (NG), Phaeodactylum tricornutum (PT)], and one heterotrophic alga [Schizochytrium sp. (SS)] were investigated. The experimental diets consisted of a hay-concentrate basal diet (BD; 200 mg dry matter) supplemented with (1) no algae (just BD), (2) 40 mg of dried NG (BD + NG), (3) 40 mg of dried PT (BD + PT), and (4) 14 mg of dried SS. In total, 48 samples (four algal treatments × two replicates × three runs × two cows) were incubated for 24 h using the Hohenheim gas test method. All three algae decreased (P < 0.05) the production of short-chain fatty acids and protozoal abundance (both adjusted in amount to BD) as compared with BD. Ammonia formation of BD + NG and BD + PT was 1.2- and 1.1-fold of values in BD, respectively. The BD + NG diet enhanced the proportions of isobutyrate, valerate, and isovalerate at cost of acetate proportion of total short-chain fatty acids, whereas the BD + PT diet promoted the proportions of propionate and valerate at cost of acetate. None of the microalgae affected in vitro methane formation. In conclusion, these algae showed a very poor fermentability and no anti-methanogenic effect in vitro.