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Objective: The objective of this study was to investigate the efficacy of second-line versus first-line antibiotics in the prevention of postoperative fever after tubeless percutaneous nephrolithotomy (PCNL). Methods: Three hundred consecutive tubeless PCNL procedures performed at our hospital between August 2010 and December 2013 were enrolled in this study. The efficacy of second-line antibiotics in the prevention PCNL-related urinary traction was reviewed by retrospective chart review. Results: The overall postoperative fever rate was 12% (36 of 300 procedures). The second-line antibiotic prophylaxis was provided in 32 procedures according to preoperative urine culture data or physician's judgment. The first-line prophylactic antibiotic was used in 268 procedures with postoperative fever in 29 procedures (10.8%). The second-line prophylactic antibiotic was used in 32 procedures with postoperative fever in seven procedures (21.9%). No significance difference of postoperative fever rate between first-line or second-line prophylactic antibiotic was found (P = 0.069). Conclusions: The second-line prophylactic antibiotic use for tubeless PCNL does not offer additional benefit over the first-line prophylactic antibiotic in the prevention of postoperative fever or shortening of the postoperative hospital stay.