Background Fever in neutropenia (FN) is a potentially life-threatening complication of chemotherapy in pediatric cancer patients. The current standard of care at most institutions is emergency hospitalization and empirical initiation of broad-spectrum antibiotic therapy. Methods We analyzed in retrospect FN episodes with bacteremia in pediatric cancer patients in a single center cohort from 1993 to 2012. We assessed the distribution of pathogens, the in vitro antibiotic susceptibility patterns, and their trends over time. Results From a total of 703 FN episodes reported, we assessed 134 FN episodes with bacteremia with 195 pathogens isolated in 102 patients. Gram-positive pathogens (124, 64%) were more common than Gram-negative (71, 36%). This proportion did not change over time (p = 0.26). Coagulase-negative staphylococci (64, 32%), viridans group streptococci (42, 22%), Escherichia coli (33, 17%), Klebsiella spp. (10, 5%) and Pseudomonas aeruginosa (nine, 5%) were the most common pathogens. Comparing the in vitro antibiotic susceptibility patterns, the antimicrobial activity of ceftriaxone plus amikacin (64%; 95%CI: 56%-72%), cefepime (64%; 95%CI 56%-72%), meropenem (64%; 95%CI 56%-72), and piperacillin/tazobactam (62%; 95%CI 54%-70%), respectively, did not differ significantly. The addition of vancomycin to those regimens would have increased significantly in vitro activity to 99% for ceftriaxone plus amikacin, cefepime, meropenem, and 96% for piperacillin/tazobactam (p < 0.001). Conclusions Over two decades, we detected a relative stable pathogen distribution and found no relevant trend in the antibiotic susceptibility patterns. Different recommended antibiotic regimens showed comparable in vitro antimicrobial activity.