Diet is the most important factor influencing the rumen microbiome, especially under long-term dietary interventions. Dietary fatty acids can affect the rumen microbiome, and this will depend on their degree of FA saturation. The objective of this research was to study the effect of supplementing the diet of dairy cows with olive oil (OO) and palm oil (HVO) on rumen microbiota. Fifteen Holstein cows averaging (±SD) 189 ± 28 d in milk were assigned to 3 treatment groups. For 63 d animals were fed a control diet (basal diet; 65% forage: 35% concentrate) with no added lipid and 2 supplemented diets (30 g/kg DM). Rumen samples were collected every 21 d for 63 d using an esophageal-ruminal sampling device. Total microbial DNA was extracted from samples for high-throughput sequencing of the 16S rRNA gene through Illumina MiSeq platform. 16S rRNA gene amplicon sequences were quality checked with FASTQC and analyzed by DADA2 and Phyloseq R packages. Taxonomy was assigned using the SILVA Database version 132. A total of 3,762,232 sequences were obtained from 48 samples and clustered into 9829 OTUs. Results revealed the dominance of phyla Firmicutes and Bacteroidetes, whereby Firmicutes was the most prevalent phylum in Control (75.2%), OO (71.1%) and HVO (75.2%). At the genus level, Succiniclasticum and Prevotella were the most dominant, and these belong to the phyla Firmicutes and Bacteroidetes, respectively. Succiniclasticum increased significantly in relative abundance post OO supplementation (P < 0.0001) throughout 63 d, while Prevotella increased (P < 0.0001) only after 42 d of OO supplementation. Following HVO dietary supplementation, Succiniclasticum increased in relative abundance (P < 0.0001) in throughout the study period, while Prevotella decreased over time (P < 0.0001). Olive oil increased the relative abundance of Succiniclasticum and Prevotella, which are related to methane mitigation and energy supply for the host, respectively. Both roles could be associated with more efficient animals and thus improved milk production. This study was sponsored by a research grant from FONDECYT 1170400, Chile.