Uterine infections are a major source of economic losses to dairy farmers. The uterine microbiota as well as opportunistic uterine contaminants can contribute to the development of endometritis in dairy cows during the postpartum period. Therefore, it is important to characterize potential pathogens and to further elucidate their role in the disease. In this study, we aimed to characterize Bacillus pumilus field isolates to obtain more details regarding their effect on uterine cells by using an in vitro endometrial epithelial primary cells model. We found that B. pumilus isolates possessed the keratinase genes ker1 and ker2 and therefore may produce keratinases. When primary endometrial epithelial cells were infected with 4 different B. pumilus strains, an effect on cellular viability was observed over the course of 72 h. The effect was dose-dependent and time-dependent. Nevertheless, significant differences between the strains were not observed. All tested strains reduced the viability of the primary cells after 72 h of incubation, indicating that B. pumilus potentially has a pathogenic effect on endometrial epithelial cells.