Background: The use of tunneled hemodialysis catheters as definitive vascular access is becoming increasingly more widespread, reaching 25% of all dialysis patients in some countries. The rate of infectious morbidity and mortality is much higher when catheters are used than when patients are dialyzed through grafts or native fistulas, and it is generally agreed that implementing appropriate preventive measures would do more to lower its incidence. Methods: A prospective, randomized, open-label, long-term follow-up clinical trial was conducted to compare the efficacy of dressing the exit site with antibiotic ointment (AO) versus catheter antimicrobial locking (AL) in preventing catheter-related bacteremia (CRB), both associated with strict standard precautions and appropriate catheter care. A total of 141 tunneled catheters, newly implanted in 116 consecutive patients, were followed during a 2-year period. Patients were randomly distributed into one of three arms, with group A receiving AO prophylaxis, group B treated with a heparin + gentamicin (5.2 mg/ml) lock (AL) and group C receiving both AO and AL prophylaxis. Results: Group A had a significantly lower infection-free time survival curve (p < 0.02, Kaplan-Meier) with a catheter survival of 103.9 days and a significantly higher number of CRB (9 episodes, p < 0.02, χ2). Group B had 130.7 mean infection-free days and 1 episode of CRB. Group C had 127.3 mean infection-free days and 5 episodes of CRB. No toxicity or other adverse events were observed during this 2-year period, and the efficacy of the preventive measures remained stable throughout. Conclusion: Antimicrobial lock is superior to AO as a CRB preventive measure. The use of lock and ointment in the same catheter was not associated with additive effects.