This study evaluated the response to 2 ruminally injected endomicrobial supplement (EMS1 and EMS2) combinations on milk composition and yield in lactating Holstein cows. The 38-d study (7-d baseline, 28-d treatment, and 10-d post-treatment period) involved 24 Holstein cows randomly allocated to 3 treatments. Animals were fed a common TMR (16.3% CP, 37.3% NDF, 0.67 Mcal of NEI/lb). Throughout the treatment period, the EMS and control treatments were directly administered to the rumen via daily injection behind the last rib in the paralumbar fossa during morning feedings. Group 1 (G1) received EMS1 containing Clostridium butyricum sp. nov. and Pichia kudriavzevii sp. nov. injected at a total of 1 × 10⁹ and 1 × 10⁹ cells/d; Group 2 (G2) received EMS2 containing C. butyricum sp. nov., P. kudriavzevii sp. nov., and Ruminococcus spp sp. nov. injected at a total of 1 × 10⁹, 1 × 10⁹, and 1 × 10⁸ cells/d; and Group 3 (G3) the control, received a basal medium suspension. Cows were milked twice daily, and milk production measurements were collected daily. Rumen tube samplings of each cow were collected on d 1, 8, 16, 24, 28, 35, and 38 to determine colonization patterns of the administered microbes via Illumina sequencing of the ITS and 16S rRNA V1-V3 hypervariable regions. All statistical comparisons of treatment main effect and 2-way interactions with treatment main effect were performed using the R package “nlme” and lme function for linear mixed models. Treatment by week interactions were observed to be significantly different for milk production (G2 vs. G3 × wk2, P = 0.0185; G2 vs. G3 × wk3, P = 0.0754), milk protein yield (G1 vs. G2 × wk2, P = 0.0302), energy-corrected milk yield (G1 vs. G3 × wk2, P = 0.0942; G2 vs. G3 × wk2, P = 0.0303), and milk protein % (G1 vs. G2 × wk5+2d, P = 0.0001; G1 vs. G3 × wk5+2d, P = 0.0009). Colonization of EMS1 and EMS2 were confirmed via integration of sequencing results with rumen content cell count data, performed using a fluorescent-activated cell sorter. These data indicate that either effective combination of EMS can have a positive effect on milk production and performance of Holstein cows.