Bacterial overgrowth in the inner layer of the catheter as a biofilm is highly encountered in routine medical care, and it may occur in a few days after inserting a catheter as an access in hemodialysis (HD) patients. Catheter-induced bacteremia is often due to the development of biofilms. Locking catheters with antimicrobial agents is an effective way of reducing the risk of catheter-related infection. In a controlled, randomized clinical trial, 64 chronic HD patients (32 men and 32 women with a mean age of 57.5 ± 15.6 years) were divided into case and control groups, with 32 patients in each group. The case group received systemic antibiotic and a lock of catheters with 60% ethanol and the control group received only systemic antibiotic. The results were evaluated after three weeks of treatment. The success rate of clearing infection in group A (29 patients) and group B (18 patients) was 90.6% and 56.2%, respectively (P = 0.002). We conclude that the significant difference in the success rate of clearing catheter infection in HD patients is due to the use of 60% ethanol-lock along with antibiotic therapy, and suggest this for routine use.