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Objective: Sepsis is the most serious complication of flexible ureteroscopy (F-URS) and laser lithotripsy. We assessed the influence of positive stone culture (SC) on major infectious complications (sepsis, septic shock). Methods: This prospective study enrolled adult patients deemed suitable for F-URS and laser lithotripsy from nine centers (January 2022–August 2023). Inclusion criteria were as follows: kidney stone(s), preoperative midstream urine culture (MSUC), stone(s) assessed at computed tomography scan, and SC. Exclusion criteria were as follows: bilateral procedures, ureteral stones, and children. Group 1 included patients with sterile SC. Group 2 included patients with positive SC. Data are presented as median (interquartile range). A multivariable logistic regression analysis was performed to evaluate factors associated with having a positive SC. Results: In total, 293 patients were included. Median age was 51.0 (24) years. There were 167 (57.0%) males. Group 2 included 32 (2.5%) patients. Group 2 patients were significantly older [75.0 (14) vs 51.0 (23) years, p = 0.02]. Stone features were similar. Major infectious complications were higher in Group 2 (15.6% vs 0.4%). One patient died because of sepsis in Group 2. Two out of 6 (33.3%) patients with major infectious complications had the same pathogen in MSUC and SC. In the multivariable regression analysis, diabetes (OR 3.23), symptomatic urinary infections within 3 months before operation (OR 4.82) and preoperative stent/nephrostomy (OR 2.92) were factors significantly associated with higher odds of positive SC. Conclusions: Patients with positive SC have a higher incidence of major infectious complications after F-URS lithotripsy. SC should be performed whenever feasible because there is a poor correlation between MSUC and SC. [ABSTRACT FROM AUTHOR]