Background: The endometrium remains a difficult tissue for the analysis of microbiota, mainly due to the low bacterial presence and the sampling procedures. Among its pathologies, endometrial cancer has not yet been completely investigated for its relationship with microbiota composition. In this work, we report on possible correlations between endometrial microbiota dysbiosis and endometrial cancer. Methods: Women with endometrial cancer at various stages of tumor progression were enrolled together with women with a benign polymyomatous uterus as the control. Analyses were performed using biopsies collected at two specific endometrial sites during the surgery. This study adopted two approaches: the absolute quantification of the bacterial load, using droplet digital PCR (ddPCR), and the analysis of the bacterial composition, using a deep metabarcoding NGS procedure. Results: ddPCR provided the first-ever assessment of the absolute quantification of bacterial DNA in the endometrium, confirming a generally low microbial abundance. Metabarcoding analysis revealed a different microbiota distribution in the two endometrial sites, regardless of pathology, accompanied by an overall higher prevalence of pathogenic bacterial genera in cancerous tissues. Conclusions: These results pave the way for future studies aimed at identifying potential biomarkers and gaining a deeper understanding of the role of bacteria associated with tumors.