The objective of this study was to compare the effect of supplementing dairy cow diets with contrasting sources of omega-6 (soybean oil) and omega-3 (fish oil) PUFA on rumen microbiome. For 63 d, 15 mid-lactating cows were fed a control diet (n=5 cows; no fat supplement) or control diet supplemented with 2.9% dry matter (DM) of either soybean oil (SO; n=5 cows) or fish oil (FO; n=5 cows). Ruminal contents were collected on days 0, 21, 42, and 63 for 16S rRNA gene sequencing. Beta diversity and Shannon, Simpson and Chao1 diversity indices were not affected by dietary treatments. In terms of core microbiome, Succiniclasticum, Prevotella, Rikenellaceae_RC9_gut_group, and NK4A214_group were the most prevalent taxa regardless of treatments. Bifidobacterium was absent in SO diet, Acetitomaculum was absent in FO, and Sharpea was only detected in SO. Overall, results showed that at 2.9% DM supplementation of either SO or FO over 63 days in dairy cow diets does not cause major impact on bacterial community composition and thus is recommended as feeding practice.