Lactobacilli with probiotic features play an essential role in maintaining a balanced vaginal microbiota and their administration has been suggested for the treatment and prevention of vaginal dysbiosis. The present study was aimed to in vitro and in vivo investigate the probiotic potential of the Lacticaseibacillus rhamnosus TOM 22.8 strain, isolated from the vaginal ecosystem of a healthy woman. For this purpose, safety and functional properties were in depth evaluated. The strain exhibited a broad spectrum of antagonistic activity against vaginal pathogens; adhesion capacity to both the vaginal VK2/E6E7 and the intestinal Caco-2 cells; anti-inflammatory and antioxidant activities, suggesting its promising probiotic features. In addition, an in vivo pilot-study was planned. Based on both clinical and microbiological parameters, the oral or vaginal strain administration, determined a significant pathogens reduction after 10 days of administration and a maintenance of eubiosis up to 30 days after the end of the treatment. Therefore, the L. rhamnosus TOM 22.8 strain can be proposed as valuable oral and/or vaginal treatment for vaginal dysbiosis.