It is generally accepted that postpartum uterine infections decrease conception rates in dairy cows. Whereas clinical endometritis (CE) has been studied intensively, only little information about CE at the time of artificial insemination (AI) is available. The aim of this study was to characterize the intrauterine cultivable aerobic microbiota in healthy cows and in cows with mild CE at AI and to investigate its effect on the subsequent insemination success. The vaginal discharge score (VDS) of 120 Holstein Frisian dairy cows was assessed shortly after AI using the Metricheck device and pregnancy diagnosis was performed 39 days after AI. On average, cows received their second insemination (2.0 ± 1.2 standard deviation (SD)) and were 120.5 ± 40.2 days in milk. Intrauterine cytobrush samples were taken from cows with clear mucus (VDS 0, n = 58) and from cows with flecks of pus (VDS 1, n = 62), which was regarded as mild CE. Bacteria collected with the cytobrush were cultivated aerobically and identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A total of 358 bacterial isolates were recovered, comprising 49 bacterial genera and 116 species. Bacillus, Staphylococcus, Corynebacterium and Streptococcus were the most prevalent genera with relative abundances of 19.6%, 14.2%, 10.1% and 8.1%, whereas common uterine pathogens, such as Trueperella pyogenes and Escherichia coli, were rarely detected. Bacillus spp. were more frequently detected in group VDS 0 (51.7%) than in VDS 1 (22.6%; P = 0.001) and there was a higher prevalence of Corynebacterium spp. in VDS 0 than in VDS 1 (37.9% vs 19.3%; P = 0.03). Cows with VDS 1 at the time of AI harbored neither Bacillus licheniformis nor Bacillus subtilis, while these species were detected in 27.6% and 8.6% of VDS 0 (P < 0.001 and P = 0.024 respectively). The insemination success in VDS 0 and 1 was 43.1 and 25.8% (P = 0.046). The diagnosis of mild CE at AI and the total bacterial load decreased the chance of pregnancy (P = 0.03) but no significant correlation between a distinctive bacterial species and insemination success was observed. In summary, cultivable uterine bacteria at AI presumably represent a part of the physiological microbiota, which is not directly linked to impaired fertility. These findings question intrauterine antimicrobial treatment shortly after AI and could represent the basis for the development of future treatment strategies.