Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides composed of repeating disaccharide units of uronic acid and amino sugars. The luminal surface of the bladder epithelium is coated with a GAG layer. These urothelial GAGs are thought to provide a protective barrier and serve as a potential interaction site with the urinary microbiome (urobiome). Previous studies have profiled urinary GAG composition in mixed cohorts, but the urinary GAG composition in postmenopausal women remains undefined. To investigate the relationship between GAGs and recurrent UTI (rUTI), we profiled urinary GAGs in a controlled cohort of postmenopausal women. We found that chondroitin sulfate (CS) is the major urinary GAG in postmenopausal women and that urinary CS was elevated in women with active rUTI. We also associated urinary GAGs with urobiome composition and identified bacterial species that significantly associated with urinary GAG concentration. Corynebacterium amycolatum, Porphyromonas somerae , and Staphylococcus pasteuri were positively associated with heparin sulfate or hyaluronic acid and bacterial species associated with vaginal dysbiosis were negatively correlated to urinary CS. Altogether, this work defines changes in urinary GAG composition associated with rUTI and identifies new associations between urinary GAGs and the urobiome that may play a role in rUTI pathobiology.