To investigate differences in the urinary microbiome and cytokine levels between women with and without interstitial cystitis and to correlate differences with scores on standardized symptom severity scales and depression and anxiety screening tools. Our cross-sectional study compared women presenting to a pelvic floor clinic and diagnosed with interstitial cystitis over a 6-month period with age-matched women in a control group from the same institution. Participants provided a catheterized urine sample and completed symptom severity, quality-of-life, depression, and anxiety screening questionnaires. Urinary microbiomes generated through bacterial ribosomal RNA sequencing and cytokine levels were analyzed using a standard immunoassay. Nonparametric analyses were used for all comparisons. Participants with interstitial cystitis reported more disability, bothersome urinary symptoms, genitourinary pain, and sexual dysfunction and scored higher on depression and anxiety screens compared with women in the control group. The urine of participants with interstitial cystitis contained fewer distinct operational taxonomic units (2 [median range 2-7, interquartile range 1] compared with 3.5 [median, range 2-22, interquartile range 5.25], P=.015) and was less likely to contain Lactobacillus acidophilus (1/14 [7%] compared with 7/18 [39%], P=.05) compared with women in the control group. L acidophilus was associated with less severe scores on the Interstitial Cystitis Symptoms Index (1 [median, range 0-17, interquartile range 5] compared with 10 [median, range 0-14, interquartile range 11], P=.005) and the Genitourinary Pain Index (0 [median, range 0-42, interquartile range 22] compared with 22.5 [median, range 0-40, interquartile range 28], P=.03). Participants with interstitial cystitis demonstrated higher levels of macrophage-derived chemokine (13.32 [median, range 8.93-17.05, interquartile range 15.86] compared with 0 [median, range 8.93-22.67, interquartile range 10.35], P=.037) and interleukin-4 (1.95 [median, range 1.31-997, interquartile range 11.84] compared with 1.17 [median, range 0.44-3.26, interquartile range 1.51], P=.029). There was a positive correlation between interleukin-4 and more severe scores on the Interstitial Cystitis Symptoms Index (r=0.406, P=.013). No associations between the presence of lactobacillus species and cytokine levels were observed. The urinary microbiome of participants with interstitial cystitis was less diverse, less likely to contain Lactobacillus species, and associated with higher levels of proinflammatory cytokines. It is unknown whether this represents causality and whether the effect of alterations to the urinary microbiome is mediated through an inflammatory response.