Unraveling the complex ecology of the vaginal biofilm microbiome relies on a number of complementary techniques. Here, we describe the experimental approaches for studying vaginal microbial biofilm samples with a focus on specimen preparation for subsequent analysis. The techniques include fluorescence microscopy, fluorescence in situ hybridization, and scanning and transmission electron microscopy. Isolation of microbial DNA and RNA from these samples is covered along with a brief discussion of chemical analysis methods.