ABSTRACT Large sex hormonal fluctuations are thought to influence vaginal microbiota, but little is known about the impact of small, physiological variations. Here we tracked changes in vaginal microbiota during four key menstrual cycle phases in 61 healthy, naturally menstruating Italian women from the Women4Health cohort. The microbiota, characterized using a high depth 16S rRNA amplicon sequencing approach covering four hypervariable regions, was primarily composed of Lactobacillus species, with Lactobacillus iners being the most abundant (average relative abundance 40%) and the most prevalent (prevalence 98%). Individual microbiota were generally stable, but beta diversity was higher during the follicular phase (p=0.007). Only 11 women exhibited compositional shifts, mostly occurring between follicular and ovulatory phases. Finally, using linear mixed models we assessed the association between taxa relative abundance and five sex-hormones along the menstrual cycle. Among these, 17-beta estradiol showed the largest number of significant associations, linking its increase to a decrease in relative abundance of taxa that are more common after menopause. Our study highlights specific features of the Italian population and points to the resilience of the vaginal microbiota to physiological hormonal changes. Noteworthy, the observed high abundance of L. iners contrasts with previous studies in European populations, challenging its proposed pathogenic role and suggesting distinct microbiota profiles within Europe. IMPORTANCE The vaginal microbiota plays an important role in women’s health, yet we know little about how it responds to normal hormonal fluctuations. In this study, we followed 61 healthy Italian women over a natural menstrual cycle to explore microbiota changes across different hormonal phases. We found that Lactobacillus iners was the most common species, unlike previous findings in Northern Europe, suggesting population-specific patterns. The common hypothesis that L. iners is invariably linked with poor health is called into question by our findings. They emphasize the importance of considering population context and hormonal status when assessing vaginal health. The vaginal microbiota was generally stable, with only a few changes observed between the follicular and ovulatory phases. When evaluating association between five sex hormones and taxa abundances, we found that 17-beta estradiol levels had the largest number of significant associations. These highlight an association between increase levels of 17-beta estradiol and increased relative abundance of rare bacterial taxa rather than dominant species like Lactobacillus. Our findings help define what constitutes a “healthy microbiota” in generally healthy Italian women of reproductive age and may inform future strategies for diagnosing or preventing women’s health conditions.