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To identify the most effective mixture of non-esterified lauric (C12) and myristic (C14) acid in suppressing ruminal methanogenesis, and to investigate their effects on the methanogenic population.C12/C14 mixtures were incubated with rumen fluid using the Hohenheim gas test apparatus. Methane production and the numbers of Archaea declined with an increasing proportion of C12. With a 2: 1 proportion of C12/C14, the maximum methane-suppressing effect (96%) was achieved similar to that with C12 alone. The proportions of the individual methanogenic orders of total methanogens were altered by varying the C12/C14 ratio.Although C14 alone had no effect on methanogenesis, C14 enhanced the methane-suppressing effect of C12 in certain mixtures.The results support strategies for an environment-friendly ruminant nutrition as it was demonstrated that part of the less palatable C12 could be replaced by C14 without losing its methane-suppressing potential.