Infections of the vaginal tract result from perturbations in the complex interactions between the microbiome and the host vaginal ecosystem. Recent data have linked specific vaginal microbes and urogenital infection with preterm birth. Here we discuss how next-generation sequencing-based approaches to study the vaginal microbiome will be important for defining what constitutes an imbalance of the microbiome and the associated host conditions that lead to subsequent infection and disease states. These studies will provide clinicians with reliable diagnostic tools and treatments for women who are at increased risk for vaginal infections, preterm birth, HIV and other sexually acquired diseases, and will provide opportunities for intervention.