Background: There is growing evidence indicating that gut microbiome content is associated with the effectiveness of immune checkpoint inhibitory (ICI) therapy in various cancer types. Antibiotic therapy (ABT) induces significant changes in gut microbiota; however, the effects of ABT on oncologic outcomes with ICI therapy has only been explored in small, single-institution studies. We aimed to investigate the relationship between ABT on overall survival (OS) and real-world progression free survival (rwPFS) in subjects with advanced renal cell carcinoma (RCC) treated with ICI utilizing a national database. Methods: We queried the nationwide electronic health record (EHR)-derived de-identified FlatIron Health (FIH) database after Institutional Review Board approval was obtained and included a waiver of informed consent to select 1809 subjects who received ICI therapy. Patient characteristics were described and compared between those with known ABT use and without known ABT use (in the 3 months pre/post ICI initiation) using ANOVA and Chi-square tests. rwPFS and OS were calculated from date of initiation of ICI to date of real-world progression/date of death. Three-month landmark Kaplan Meier and log-rank tests were used to compare rwPFS and OS between groups defined by ABT use (any ABT, and ABT in 3 months pre-ICI vs 3 months post-ICI initiation). Cox proportional models were used to investigate the association between rwPFS, OS, and antibiotic use, adjusting for patient characteristics. Results: Patients receiving ABT group were younger (p = 0.007) and more likely female (p = 0.024) but similar in other measured demographic variables. rwPFS (median: 9.03 vs 11.6 months; p = 0.015) and OS (median: 23.7 vs 31.8 months; p = 0.019) were significantly shorter in the with-ABT group than in the without-ABT group. When assessing ABT use 3 months prior to initiation of ICI (vs ABT during/no known ABT use), neither rwPFS (median: 9.53 vs 11.66, p = 0.38) nor OS (median: 23.7 vs 22.6 months; p = 0.38) were significantly different. However, rwPFS (median: 23.5 vs 14.0 months vs not reached; p = 0.004) and OS (median: 36.2 vs 22.5 vs 31.8; p = 0.007) were statistically significantly different when stratifying based on exposure and timing of ABT amongst pre-ICI initiation ABT, post-ICI initiation ABT and no ABT groups, respectively. Multivariable analysis identified as a significant predictor of rwPFS (HR = 0.725, CI = 0.579-0.908; p =.005) and OS (HR = 0.722, CI = 0.573-0.911; p = 0.006). Conclusions: This study identifies a significant negative prognostic association of ABT administration on survival in advanced RCC treated by ICI in the first line setting. These results are consistent with the growing body of evidence suggesting that alterations in gut microbiome secondary to antibiotics appear to blunt efficacy of ICI treatment and emphasize the importance of antibiotic stewardship.