Background: Pancreatic cystic lesions present a challenge for patients and physicians alike. Morphology alone is inaccurate in discriminating lesion pathology, and use of EUS-guided FNA (EUS-FNA) improves accuracy. Current American Society for Gastrointestinal Endoscopy guidelines recommend prophylactic antibiotics during FNA of cystic lesions to minimize infection risk. However, evidence pertaining to infection risk has been conflicting. The use of prophylactic antibiotics might not be free of other adverse events and might not prevent infection. Objective: To assess the impact of antimicrobial therapy for prophylaxis during EUS-FNA of pancreatic cysts. Design: Retrospective cohort study. Patients: This study involved all patients who underwent EUS-FNA of pancreatic cysts at one institution from May 2007 to April 2010. Intervention: Antibiotic prophylaxis for EUS-FNA. Main Outcome Measurements: Infection of a pancreatic cyst, fever, or bacteremia after EUS-FNA. Secondary variables included other complications of the procedure related to the use of prophylaxis (ie, allergic reactions, secondary infections). Results: EUS-FNA was performed on 253 patients in 266 procedures. Antibiotics were used in 88 endoscopy cases (ATB group), whereas no antibiotics were used in 178 cases (NATB group). There were no differences in patient or cyst characteristics between groups. There were 4 major complications in the NATB group (localized bleeding, 2; pancreatitis, 1; bile leakage, 1) and 2 in the ATB group (possible cyst infection, 1; bile leakage, 1) (P = 1.0). Eight mild adverse events were observed in the NATB group and 6 in the ATB group (P = .56). Infections and antibiotic-related complications occurred in 1 (0.6%) (transient fever) in the NATB group and 4 (4.5%) in the ATB group (local allergic reaction, 2; possible cyst infection, 1; Clostridium difficile diarrhea, 1) (P = .04). Limitations: Retrospective analysis. Conclusion: The incidence of infectious complications after EUS-FNA of pancreatic cystic lesions, with or without antibiotic prophylaxis, appears very low. We have not observed a protective effect from periprocedural prophylactic antibiotic administration. © 2011 American Society for Gastrointestinal Endoscopy.