Recent molecular studies concluded that the endometrium has a resident microbiota dominated by Lactobacillus spp. and is therefore similar to that of the vagina. These findings were largely derived from endometrial samples obtained through a transcervical catheter and thus prone to contamination. Herein, we investigated the molecular microbial profiles of mid-endometrial samples obtained through hysterectomy and compared them with those of the cervix, vagina, rectum, oral cavity, and controls for background DNA contamination. Microbial profiles were examined through 16S rRNA gene qPCR and sequencing. Universal bacterial qPCR of total 16S rDNA revealed a bacterial load exceeding that of background DNA controls in the endometrium of 60% (15/25) of the study subjects. Bacterial profiles of the endometrium differed from those of the oral cavity, rectum, vagina, and background DNA controls, but not of the cervix. The bacterial profiles of the endometrium and cervix were dominated by Acinetobacter, Pseudomonas, Cloacibacterium, and Comamonadaceae. Both 16S rRNA gene sequencing and Lactobacillus species-specific (L. iners & L crispatus) qPCR showed that Lactobacillus was rare in the endometrium. In conclusion, if there is a microbiota in the middle endometrium, it is not dominated by Lactobacillus as was previously concluded, yet further investigation using culture and microscopy is necessary.