We aimed to describe the epidemiology of catheter-related bloodstream infections (CRBSIs) in onco-hematological neutropenic patients during a 25-year study period, to evaluate the risk factors for Gram-negative bacilli (GNB) CRBSI, as well as rates of inappropriate empirical antibiotic treatments (IEAT) and mortality. All consecutive episodes of CRBSIs were prospectively collected (1994-2018). Changing epidemiology was evaluated comparing five-year time spans. A multivariate regression model was built to evaluate risk factors for GNB CRBSIs. 482 monomicrobial CRBSIs were documented. The proportion of CRBSIs among all BSIs decreased over time from 41.2% to 15.8% (p<0.001). CRBSIs epidemiology has been changing: the rate of GNB increased over time (from 11.9% to 29.4%; p<0.001), as well as the absolute number and rate of multidrug-resistant (MDR) GNB (from 9.5% to 40.0%; p = 0.039). P. aeruginosa increased and comprised up to 40% of all GNB. Independent factors related with GNB-CRBSIs were: longer duration of in-situ catheter (OR 1.007; 95%CI 1.004-1.011), older age (OR 1.016; 95%CI 1.001-1.033), prior antibiotic treatment with penicillins (OR 2.716; 95%CI 1.306-5.403), and current antibiotic treatment with glycopeptides (OR 1.931; 95%CI 1.001-3.306). IEATs were administered to 30.7% of patients, with the highest percentage among MDR P. aeruginosa (76.9%) and S. maltophillia (92.9%). Mortality rate was greater among GNB than GPC-CRBSI (14.4% vs 5.4%; p = 0.002), with mortality increasing over time (from 4.5% to 11.2%; p = 0.003). A significant shift towards GNB-CRBSIs was observed. Secondarily, and coinciding with an increasing number of GNB-MDR infections, mortality increased over time.