Background: Standard therapy for high-risk non-muscle invasive bladder cancer (NMIBC) is intravesical BCG. Despite its established efficacy, up to 50% of patients may recur. The development of predictive biomarkers for BCG immunotherapy would enhance treatment algorithms and potentially uncover mechanisms of resistance. Recent data have characterized the presence of a commensal urine microbiome, but its role in BCG-treated NMIBC patients remains unexplored. We assessed the composition of the urine microbiome in NMIBC patients and evaluated associations with response to therapy. Methods: Patients with a newly diagnosed bladder tumor were enrolled prior to TURBT on an institutional review board-approved study at a single institution. Adjuvant BCG instillation was administrated to high-risk patients according to the surgical histology. Urine samples for microbiome assessment were collected by catheterization to eliminate urethral contamination before the TURBT and up to 8 additional timepoints. 16S sequencing and analysis was performed on baseline specimens prior to BCG. Results: Among 31 patients enrolled, 10 (32%) recurred and 21 (68%) had no recurrence with a median follow up of 6 months. Median age was 69 years (range 46-87), and 9 (29%) patients were female and 22 patients (71%) were male. The most abundant phyla observed were Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Tenericutes. Together these accounted for > 99% of the phyla detected in the cohort. Global analysis of distances by operational taxonomic units (OTUs) indicated a significant difference between patients with and without recurrence (Bonferroni-corrected P = 0.017). The abundance of Proteobacteria was higher in patients with recurrence (P = 0.035), with stronger differences observed for specific classes such as Gammaproteobacteria (P = 0.0025). Firmicutes such as Lactobacillales were more abundant in patients without recurrence (P = 0.049). Conclusions: In this study we detected variation in the composition of the urine microbiome in NMIBC patients, which may predict response to BCG immunotherapy. Further studies to confirm these results are ongoing.