Catheter-associated urinary tract infection (CAUTI) remains the most significant challenge among hospital-acquired infections (HAIs), yet still unresolved. The present study aims to evaluate the preventive effectiveness of JUC Spray Dressing (name of U.S. FDA and CE certifications, while the medical device name in China is Long-acting Antimicrobial Material) alone for CAUTI without combining with antibiotics and to evaluate the impact of bacterial biofilm formation on CAUTI results on the inserted catheters of patients. In this multicenter, randomized, double-blind study, we enrolled adults who suffered from acute urinary retention (AUR) and required catheterization in 6 hospitals in China. Participants were randomly allocated 1:1 according to a random number table to receive JUC Spray Dressing (JUC group) or normal saline (placebo group). The catheters were pretreated with JUC Spray Dressing or normal saline respectively before catheterization. Urine samples and catheter samples were collected after catheterization by trial staff for further investigation. From April 2012 to April 2020, we enrolled 264 patients and randomly assigned them to the JUC group (n = 132) and the placebo group (n = 132). Clinical symptoms and urine bacterial cultures showed the incidence of CAUTI of the JUC group was significantly lower than the placebo group (P < 0.01). In addition, another 30 patients were enrolled to evaluate the biofilm formation on catheters after catheter insertion in the patients' urethra (10 groups, 3 each). The results of scanning electron microscopy (SEM) showed that bacterial biofilm formed on the 5th day in the placebo group, while no bacterial biofilm formed on the 5th day in the JUC group. In addition, no adverse reactions were reported using JUC Spray Dressing. Continued indwelling urinary catheters for 5 days resulted in bacterial biofilm formation, and pretreatment of urethral catheters with JUC Spray Dressing can prevent bacterial biofilm formation by forming a physical antimicrobial film, and significantly reduce the incidence of CAUTI. This is the first report of a study on inhibiting bacterial biofilm formation on the catheters in CAUTI patients.