<b><i>Introduction:</i></b> The incidence of catheter-related bloodstream infections (CRBSI) ranges from 2.2 to 5.5 episodes per 1,000 catheter-days. Our aim was to evaluate the utility of a generalized and prolonged gentamicin-lock therapy in patients undergoing hemodialysis (HD) in a third-level hospital for the reduction in CRBSI. <b><i>Methods:</i></b> A prospective cohort analyzed before and after intervention. During intervention periods after each HD-session, the catheter lumens were locked with gentamicin/heparin for all patients compared to nonintervention periods were the same procedure was performed without gentamicin. Active surveillance was performed for HD CRBSI. Microbiologic assessment and epidemiological data were gathered. Continuous hand hygiene and water quality monitoring were performed. <b><i>Results:</i></b> The rates of CRBSI were reduced from 1.28 to 0.2 cases per 1,000 catheter-days when the lock therapy was employed (<i>p</i> = 0.001) The greatest reduction was for CRBSI caused by <i>Pseudomonas aeruginosa</i> were no cases were recorded during the intervention periods (<i>p</i> = 0.001). There was a significant reduction in the total number of isolates; Gram-negative bacterial species (–97.2%) and Gram-positive bacterial species (–61.5%) although only the former reached statistical significance (<i>p</i> = 0.0001). The difference in the absolute risk reduction was 20.56% (95% CI 14.46–26.66%), the calculated Number Needed to Treat was 5 (95% CI 3.8–6.9). No adverse effects were noted. <b><i>Conclusion:</i></b> In the current study, gentamicin-lock therapy was associated with a significant reduction in CRBSI specially with <i>P. aeruginosa</i> and other Gram-negative bacteria. It proved to be safe and effective intervention when applied to the entire population of HD patients.