Background: Use of Abx may negatively affect outcomes with ICI, possibly due to negative impact on gut microbiota. We previously showed that in our real world cohort of 138 mUC pts, Abx use within 30 or 60 days (d) of ICI initiation did not impact overall survival (OS). We now report updated follow-up (fu) and results from our expanded cohort of 310 mUC pts who received ICI. Methods: We performed a retrospective analysis of adult pts with mUC treated at the Cleveland Clinic between 2015 and 2022 who received 2 or more cycles of ICI with pembrolizumab (P), atezolizumab (A) or avelumab (Av). Effect of Abx use within 30 and 60 (d) of ICI initiation was correlated with progression free survival (PFS) and OS. OS and PFS were estimated using Kaplan-Meier method and compared using log rank test. Results: Median age of pts was 71 years (41-90) and 75% pts were males. 224 pts (72.3%) received P, 82 (26.4%) A and 4 (1.3%) Av. Fluoroquinolones (31.1%), penicillins (29.6%) and cephalosporins (19.7%) were most commonly used Abx. With a median fu of 47 weeks (3.86- 325.3), median OS in pts who received Abx within 60 (d) prior to ICI was 11.79 months (mos) vs 14.55 mos in those who did not receive Abx (p=0.007) and median PFS was 3.68 mos vs 5.29 mos respectively (p=0.038). Median OS in pts who received Abx within 30 days prior ICI was 10.41 mos vs 14.46 mos in those who did not receive Abx (p=0.037). There was a non-significant trend for worse PFS in pts who received Abx within 30 (d) prior to ICI (3.06 mos vs 5.03 mos, p=0.238). There was no effect on OS or PFS with Abx use 30 or 60 (d) after ICI initiation. Conclusions: In our real-world cohort of mUC pts receiving ICI, Abx use 60 and 30 (d)prior to ICI initiation was associated with significant worse OS and worse PFS for the former group but Abx use after ICI initiation did not have an effect on OS or PFS. These findings have the potential to influence clinical practice, including a higher threshold for giving Abx to mUC pts starting ICI. Further prospective studies are warranted.