Background: Preoperative antibiotic prophylaxis is commonly used to reduce surgical site infections (SSIs). However, the rate of SSIs following surgical procedures classified as clean is only 2-3%. Overuse of antibiotics is associated with several potential adverse effects, including dysregulation of the gut microbiome. Disruption of the composition and function of the native gut microbiota, referred to as dysbiosis, has been implicated in a number of inflammatory and autoimmune disorders, as well as gastrointestinal (GI) and non-GI cancers. Recent studies have demonstrated that antibiotics have a profound and persistent effect on the gut microbiota, as evidenced by diminished overall abundance and diversity, as well as alteration of community composition that includes a decreased relative abundance of bacteria in the Ruminococcaceae family. In melanoma, diversity of gut microbiota and relative abundance of Ruminococaceae have been linked to improved survival and enhanced response following immune checkpoint blockade. In this study, we seek to determine the impact of preoperative prophylactic antibiotic use on the gut microbiome in patients following surgery for stage I or II melanoma. Methods: In this non-comparative randomized pilot trial, the impact of prophylactic antibiotic use at the time of surgical intervention on gut microbiome diversity and composition will be studied. Patients diagnosed with clinical stage I or II melanoma undergoing wide excision with or without lymphatic mapping and sentinel lymph node biopsy are randomized 1:1 to either receive preoperative cefazolin or no preoperative antibiotics. Stool samples and peripheral blood are collected before surgery, the day of surgery (optional), on post-operative day 3 (optional), and 2 weeks and 3 months following surgery. The primary endpoint for the study is change in microbiome alpha diversity at 2 weeks following surgery. Secondary endpoints are change in relative abundance of microbes at 2 weeks and 3 months after surgery and SSI rates according to whether or not prophylactic antibiotics were administered at time of surgery. Exclusion criteria include recent antibiotic use (within 3 months), allergy to beta-lactam or cephalosporin antibiotics, increased risk of infection due to medical comorbidity or use of immunosuppressive medication. Enrollment began in October 2021. As of January 2022, 22 of 30 patients have been accrued to ensure complete sample collection for 20 patients. Study findings may inform a larger trial evaluating interventions to mitigate antibiotic impact. Clinical trial information: NCT04875728.