Catheter-related bacteremia (CRB) is a typical complication of hemodialysis catheter use. Catheter lumen colonization by pathogens is regarded as a direct cause of CRB. Once settled, the catheter biofilm increases the risk of developing infection, thus necessitating insertion replacement and antibiotic treatment. The study assessed the self-sufficient efficacy of taurolidine–citrate–heparin lock solution in eradicating catheter biofilm bacteria and keeping it sterile in patients on hemodialysis. Twenty-nine chronic patients on hemodialysis with tunneled and nontunneled catheters locked with a heparin filling (the mean time of heparin lock use −30.1 ± 2.0 days) and subsequently converted to a taurolidine–citrate–heparin filling were included. Peripheral vein and catheter lumen blood cultures were obtained before the filling change and after taurolidine–citrate–heparin lock use (mean time 33.8 ± 7.6 days). Twenty-four participants with tunneled and nontunneled catheters locked with taurolidine–citrate–heparin filling served as the control group. During the heparin-locking period, CRB was diagnosed in 3 cases (only nontunneled catheters). The catheter blood cultures findings were positive in 23 patients (10 temporary and 13 permanent catheters), whereas both the catheter and peripheral vein blood cultures were sterile in 3 of 29 subjects (only permanent catheters). Irrespective of catheter type (tunneled or nontunneled), repeated culture revealed no pathogens in any of the 23 patients with initial positive catheter blood culture, after the use of taurolidine–citrate–heparin filling. No positive blood culture was noted in the control group. The taurolidine–citrate–heparin lock solution effectively eradicated pathogens from nontunneled and tunneled catheter biofilms and helped to maintain catheter lumen sterility.