We assessed and ranked different dietary strategies for mitigating methane (CH4) emissions and other fermentation parameters, using an automated gas system in two in vitro experiments. In experiment 1, a wide range of dietary CH4 mitigation strategies was tested. In experiment 2, the two most promising CH4 inhibitory compounds from experiment 1 were tested in a dose-response study. In experiment 1, the chemical compounds 2-nitroethanol, nitrate, propynoic acid, p-coumaric acid, bromoform, and Asparagopsis taxiformis (AT) decreased predicted in vivo CH4 production (1.30, 21.3, 13.9, 24.2, 2.00, and 0.20 mL/g DM, respectively) compared with the control diet (38.7 mL/g DM). The 2-nitroethanol and AT treatments had lower molar proportions of acetate and higher molar proportions of propionate and butyrate compared with the control diet. In experiment 2, predicted in vivo CH4 production decreased curvilinearly, molar proportions of acetate decreased, and propionate and butyrate proportions increased curvilinearly with increased levels of AT and 2-nitroethanol. Thus 2-nitroethanol and AT were the most efficient strategies to reduce CH4 emissions in vitro, and AT inclusion additionally showed a strong dose-dependent CH4 mitigating effect, with the least impact on rumen fermentation parameters.