Background: Immune checkpoint inhibitors (ICIs), specifically anti-PD-1 therapy, have improved survival in advanced-stage melanoma patients compared to previous treatment options. A healthy gut microbiome has been associated with a positive response to ICIs. Antibiotics are known to disrupt the gut microbiome and may have immune-modulatory effects within the tumor microenvironment. Studies have demonstrated a negative impact on survival with any antibiotic use during ICI treatment. Less information is known regarding the impact of spectrum of activity or antibiotic timing on survival and risk of ICI-associated gastritis/colitis. Methods: We conducted a single-center, retrospective review of advanced-stage melanoma patients treated with anti-PD-1-based therapy (ipilimumab/nivolumab or anti-PD-1 monotherapy) between 2014-2022. Multiple clinicopathologic variables including antibiotic use, survival outcomes, and development of colitis and/or gastritis were assessed. Antibiotic spectrum of activity was divided into two groups: with anaerobic coverage and without anaerobic coverage. Antibiotic timing was assessed as patients who received antibiotics within 30-days prior to starting ICI and/or during treatment. Univariate and multivariate analyses on PFS and OS were performed using Cox proportional hazard models. Logistic regression models were used to assess risk of gastritis and/or colitis. Multivariate analyses accounted for age, gender, melanoma primary type, BRAF mutation status, presence of brain or liver metastases, and pre-treatment LDH levels. Results: A total of 214 patients were analyzed. Multivariate analysis demonstrated any antibiotic exposure during anti-PD-1 therapy was associated with improved OS [HR 0.500, 95% CI 0.303–0.826, p = 0.007]. Although not statistically significant, there was a trend towards worse OS in patients who received antibiotic therapy 30-days prior to ICI therapy [HR 1.726, 95% CI 0.994-2.995, p = 0.052]. Use of antibiotics without anaerobic coverage was associated with improved PFS [HR 0.462, 95% CI 0.273–0.782, p = 0.004] and OS [HR 0.399, 95% CI 0.200–0.789, p = 0.009]. Antibiotic exposure 30-days prior to initiating anti-PD-1 therapy increased the risk of developing gastritis and/or colitis [OR 1.990, 95% CI 0.744–2.651, p = 0.043]. Conclusions: Antibiotic timing and spectrum of activity may be predictive of survival outcomes and risk of gastritis/colitis in anti-PD-1-treated patients with advanced-stage melanoma. Unlike previous studies, we found improved survival in patients receiving antibiotics during treatment and in those receiving antibiotics without anaerobic coverage. Our results demonstrate the need to further investigate specific antibiotic agents as well as antibiotic timing and their clinical impact on patients receiving ICI treatment.