The human vagina hosts a diverse ecosystem of microorganisms, including bacteria, viruses, and fungi, which play crucial roles in maintaining women's and fetal health. The study establishes the Vaginal Microbial Genome Collection (VMGC), a comprehensive resource comprising 33,804 microbial genomes from 786 prokaryotic species, 11 fungal species, and 4,263 viral operational taxonomic units (vOTUs). Notably, over 25% of prokaryotic species and 85% of vOTUs remain uncultured. The VMGC significantly enriches genomic diversity, particularly for prevalent vaginal pathogens such as BVAB1 and Amygdalobacter spp. The collection enables the characterization of functional traits of prokaryotes, including Saccharofermentanales, and prokaryotic and eukaryotic viruses, providing insights into their niche adaptation and potential roles in the vagina. The VMGC serves as a valuable resource for studying the vaginal microbiota and its impact on vaginal health. The study also highlights the need for further research to understand the functional contributions of uncultured microorganisms and the potential implications for disease development.The human vagina hosts a diverse ecosystem of microorganisms, including bacteria, viruses, and fungi, which play crucial roles in maintaining women's and fetal health. The study establishes the Vaginal Microbial Genome Collection (VMGC), a comprehensive resource comprising 33,804 microbial genomes from 786 prokaryotic species, 11 fungal species, and 4,263 viral operational taxonomic units (vOTUs). Notably, over 25% of prokaryotic species and 85% of vOTUs remain uncultured. The VMGC significantly enriches genomic diversity, particularly for prevalent vaginal pathogens such as BVAB1 and Amygdalobacter spp. The collection enables the characterization of functional traits of prokaryotes, including Saccharofermentanales, and prokaryotic and eukaryotic viruses, providing insights into their niche adaptation and potential roles in the vagina. The VMGC serves as a valuable resource for studying the vaginal microbiota and its impact on vaginal health. The study also highlights the need for further research to understand the functional contributions of uncultured microorganisms and the potential implications for disease development.