This study evaluated the effects of different dietary n-6/n-3 polyunsaturated fatty acid (PUFA) ratios on in vitro ruminant fermentation. Methane production, fatty acid composition, and microbial profiles were compared after the in vitro fermentation of rumen fluid collected from cows that had been fed isoenergetic and isoproteic experimental diets at three different n-6/n-3 ratios: 3.04 (HN6, high n-6 source), 2.03 (MN6, medium n-6 source), and 0.8 (LN6, low n-6 source). The fermented rumen fluid pH and total volatile fatty acid (VFA) levels were significantly decreased (p < 0.05) in the HN6 group as compared with those in the MN6 and LN6 groups. Additionally, the HN6 group produced a significantly lower (p < 0.05) proportion of methane than the MN6 group during in vitro fermentation. The MN6 and LN6 groups had significantly increased (p < 0.05) levels of C18:2n6 and C18:3n3 in the fermented rumen fluid, respectively, as compared with the HN6 group. The Chao 1 diversity index value was lower (p < 0.05) in the HN6 group than in the MN6 and LN6 groups. The observed species richness was significantly lower (p < 0.05) in the HN6 group than in the MN6 group. The reduced relative abundances of Lachnospiraceae UCG-006 and Selenomonas in the HN6 group resulted in lower pH and VFA levels (i.e., acetate, propionate, butyrate, and total VFA) during in vitro fermentation. Furthermore, n-6 and n-3 PUFAs were toxic to Butyrivibrio_2 growth, resulting in high levels of incomplete biohydrogenation. Taken together, the study findings suggest that supplementation of high-forage diets with high levels of n-6 PUFAs could reduce methane emissions, whereas both VFA concentration and pH are reduced.