Endometritis is one of the most important causes of infertility in dairy cows, resulting in high economic losses in the dairy industry. Though the presence of a commensal uterine microbiota is now well established, the complex role of these bacteria in genital health, fertility, and susceptibility to uterine diseases remains unclear. In this study, we explore the endometrial microbiota through 16S rRNA gene profiling from cytobrush samples taken ex vivo from healthy, pregnant, and endometritis cows. There were no significant differences between healthy and pregnant cows, whose uterine microbiota were dominated by Streptococcus, Pseudomonas, Fusobacterium, Lactococcus and Bacteroides. Compared to pregnant and clinically healthy cows, the uterine bacterial community of endometritis cows was significantly decreased in species diversity (p < 0.05), reflecting uneven community composition in different patterns with either dominance of Escherichia-Shigella, Histophilus, Bacteroides and Porphyromonas or Actinobacteria.