Objective <p>To evaluate the efficacy and safety of fosfomycin tromethamine powder (FMT) for preventing biofilm-forming bacterial colonization on double-J (DJ) stents in patients with upper urinary tract calculi.</p> Methods <p>From January 2024 to June 2025, we prospectively enrolled 200 adult patients with upper urinary tract calculi undergoing double-J stent placement after endoscopic laser lithotripsy at our tertiary medical center and randomly assigned them in a 1:1 ratio to the experimental or control group (<i>n</i> = 100 per group). Patients in the experimental group received a single oral dose of 3&#xa0;g FMT one day before double-J stent placement and on postoperative days 7 and 15. Patients in the control group received intravenous cefuroxime (CXM), 1.5&#xa0;g per dose, administered twice daily one day before stent placement and on postoperative days 1 and 2. Stent surface samples were collected at the time of DJ stent removal for quantitative bacterial biofilm culture, microbial species identification, and antimicrobial susceptibility testing. Baseline clinical characteristics, Double-J Stent Symptom Score (DJSS) (assessed during indwelling), SF-36 Health Survey quality-of-life score, numerical rating scale (NRS) pain score, and urine analysis parameters at stent removal were systematically recorded. All statistical analyses were conducted using SPSS version 26.0, with statistical significance defined as a two-sided <i>p</i>-value &lt; 0.05.</p> Results <p>No statistically significant differences were observed in baseline characteristics between the experimental and control groups. Biofilm-forming bacterial colonization was identified in 49 individuals (24.5%). The colonization rate was significantly lower in the experimental group (17/100, 17%) than in the control group (32/100, 32%) (χ² = 6.082, <i>P</i> = 0.014). The experimental group demonstrated statistically significant improvements over the control group in preventing ureteral stent syndrome, reducing postoperative pyrexia incidence, decreasing urine leukocyte esterase positivity rate, lowering urinary leukocyte count, alleviating postoperative pain (assessed using a numerical rating scale), and improving SF-36 health survey quality-of-life scores (all <i>P</i> &lt; 0.05). The three most frequently isolated pathogens were <i>Escherichia coli</i> (<i>E. coli</i>) (<i>n</i> = 17, 34.7%), <i>Staphylococcus aureus</i> (<i>n</i> = 8, 16.3%), and <i>Klebsiella pneumoniae</i> (<i>n</i> = 5, 10.2%). Antimicrobial susceptibility testing demonstrated that Gram-negative bacilli exhibited high-level resistance to ampicillin, ciprofloxacin, levofloxacin, cefazolin, cefuroxime, and ceftriaxone. In contrast, these isolates remained highly susceptible to β-lactam/β-lactamase inhibitor combinations (cefoperazone/sulbactam and piperacillin/tazobactam), carbapenems (meropenem, imipenem, and ertapenem), cefotetan, nitrofurantoin, and fosfomycin. Gram-positive cocci exhibited high-level resistance to oxacillin, penicillin G, cefoxitin, and erythromycin. Conversely, these isolates demonstrated consistently high susceptibility to linezolid, vancomycin, tigecycline, and rifampicin. Fungal isolates showed high susceptibility to itraconazole, fluconazole, voriconazole, 5-fluorouracil, and amphotericin B.</p> Conclusion <p>When both groups received a fixed 3-day drug regimen, Compared to CXM, FMT may reduce the proportion of upper urinary tract calculi patients with biofilm-forming bacteria on DJ stents in this region. These findings support FMT—rather than CXM—as the preferred agent for targeted prophylaxis in this region-specific patient population and underscore the importance of rational antibiotic use. Optimizing infection prevention strategies requires integration of antimicrobial susceptibility test results with population-specific clinical characteristics, including established infection risk factors.</p>