Background and objectives <p>The vaginal microbiome plays a critical role in reproductive health through Lactobacillus-dominated communities that maintain acidic pH and suppress pathogens. Disruptive hygiene practices, such as douching and certain feminine products, may negatively affect this balance. Limited data exist on vaginal microbiome awareness and related behaviors among women in Saudi Arabia. This study aimed to assess women’s knowledge of the human and vaginal microbiome and examine associated hygiene practices.&#xa0;</p> Materials and methods <p>A cross-sectional online survey was conducted between November 2024 and February 2025 among women aged ≥ 18 years residing in Saudi Arabia. A total of <i>N</i> = 1,040 participants were recruited using convenience and snowball sampling through social media platforms. The questionnaire assessed sociodemographic characteristics, microbiome knowledge, and vaginal hygiene practices. Associations were examined using multivariable linear regression, and results are reported with 95% confidence intervals (CIs).&#xa0;</p> Results <p>Participants demonstrated moderate general microbiome knowledge but limited understanding of the vaginal microbiome. Only 20.67% correctly identified <i>Lactobacillus</i> as the dominant genus in a healthy vaginal ecosystem. Higher knowledge scores were observed among women with higher education and those working in healthcare. Use of internal vaginal washes was significantly associated with lower vaginal microbiome knowledge (β = − 0.30, 95% CI: − 0.58 to − 0.01, <i>p</i> = 0.0403). Other feminine product use showed no significant associations with knowledge levels.&#xa0;</p> Conclusions <p>Substantial gaps exist in vaginal microbiome awareness among Saudi women, accompanied by frequent use of potentially disruptive hygiene practices. Targeted health education and culturally appropriate interventions are needed to promote microbiota-supportive behaviors and improve reproductive health outcomes.</p>