We have developed a bladder-specific bacterial reference database consisting of 1134 genomes to aid in genomic, functional, and experimental analyses of the human bladder microbiota. The genomes were derived from bacterial isolates collected from bladder urine through transurethral catheterization using a metaculturomic approach. The reference database encompasses 196 species, including major aerobes, facultative anaerobes, and some anaerobes. It will facilitate bladder microbiota research by allowing hypothesis-driven investigations and cross-site comparisons.