This study was part of a bloodstream infection surveillance programme that prospectively collected data on consecutive patients with bacteraemia in our institution from 1991 to 2012. We included 2092 bacteraemias in neutropenic patients. Shock and mortality accounted for 299 and 349 cases, respectively (14% and 17%). The main microorganisms isolated were coagulase-negative staphylococci (CoNS, 634, 30%), Escherichia coli (468, 22%) and Pseudomonas aeruginosa (235, 11%). During 2006–2012, there were 155 (27%) E. coli isolates; of these, 73% were fluoroquinolone resistant and 26% cefotaxime resistant. The independent risk factors for mortality were shock on presentation, rapidly fatal prognosis of underlying disease, corticosteroid use, and polymicrobial bacteraemia. Factors associated with lower mortality were the isolation of CoNS [odds ratio (OR) 0·38, 95% confidence interval (CI) 0·20–0·73, P = 0·004] and empirical therapy with amikacin (OR 0·50, 95% CI 0·29–0·88, P = 0·016). The progressive increase of Gram-negative microorganisms resistant to antibiotics influences the choice of empirical treatment in febrile neutropenia and in our experience, the addition of amikacin could be beneficial for such patients.