Effect of an endomicrobial supplement (EMS) on dairy cow milk composition and yield was assessed. The EMS consisted of Clostridium butyricum sp. nov. and Pichia kudriavzevii sp. nov., injected at a total of 4 × 10⁹ and 1 × 10⁹ cells/day. Observations were collected from 16 multiparous, ruminally cannulated Holstein cows that were randomly split into a control (CON) and inoculated (INO) group. Study consisted of 3 periods: 10-d pre-treatment, 32-d treatment, and 10-d post-treatment. Cows were individually penned and fed a common TMR (17% CP, 27.1% NDF) twice daily. During morning feedings of TRT, INO received the EMS and CON received sterile PBS via rumen cannula. A composite milk sample per cow was collected at each milking on d 10 pre-TRT, and daily during the TRT and post-TRT periods. Milk composition was analyzed using near-infrared spectroscopy for crude protein, fat, and milk urea nitrogen (MUN) at the Tulare DHIA Laboratory. Data were analyzed by averaging daily values to produce weekly means for conducting repeated measures using the MIXED procedure of SAS. A composite rumen fluid sample was collected 18 times throughout the 52-d study to determine EMS colonization by sequencing the ITS and 16S rRNA V1-V3 hypervariable regions on the Illumina MiSeq Platform. EMS abundance of INO, compared with the CON, had increased on d 2 of TRT. Peak relative abundance of C. butyricum sp. nov. (1.4%) and P. kudriavzevii sp. nov. (5%) occurred at d 19 in INO. A tendency for a higher milk fat percentage for INO vs. CON group was observed (P = 0.0991). A treatment by week interaction was observed for milk yield (P = 0.0025), fat-corrected milk (FCM, P = 0.0026), energy-corrected milk (ECM, P = 0.0019), protein yield (PY, P = 0.0012), fat yield (FY, P = 0.0880), feed efficiency (FE, P = 0.0671) and rumen pH (P = 0. 0741). Though treatment by week interaction was not significant, results indicate that under the conditions of this study, EMS containing Clostridium butyricum sp. nov. and Pichia kudriavzevii sp. nov., may have a positive effect on cow performance.