The human microbiome varies according to individual and geographic characteristics. The endometrial microbiome plays a relevant role in reproductive physiology; however, data from healthy South American populations remain scarce. This prospective observational study aimed to characterize the endometrial microbiome in healthy reproductive-aged women from Rosario, Argentina and to establish a regional baseline for future research. A total of 100 healthy women aged 22-50 years from Rosario, Argentina, were included. Endometrial samples were obtained by aspiration biopsy during the follicular phase of the menstrual cycle or under hormonal contraception. Microbiome analysis was performed using 16S rRNA gene sequencing targeting the V4 region on the Illumina NextSeq 500/550 platform (2×150bp). Bioinformatic analysis was carried out in QIIME2. Of the 98 samples successfully amplified, 86 met quality criteria and were included in the final analysis. The endometrial microbiome was predominantly composed of Firmicutes, particularly members of the Lactobacillaceae family. The most abundant species were Lactobacillus iners and Lactobacillus helveticus/crispatus. No significant differences in alpha diversity were observed among age groups; however, a progressive increase in Gardnerella, Prevotella, and Megasphaera was detected with advancing age. This study provides the first regional characterization of the normal endometrial microbiome in healthy Argentine women and establishes a reference framework for future studies addressing endometrial dysbiosis and reproductive disorders.