Episodes of fever and neutropenia are common complications of treatment for cancer. The use of prophylactic and early empirical antibiotics has reduced mortality but decreases the sensitivity of diagnostic tests based on culture. The aim of this study was to determine the potential of a broad diagnostic approach (eubacterial) based on 16S rRNA gene amplification and sequencing to augment cultural methods of diagnosis of bacteraemia in patients with fever and neutropenia in a regional paediatric oncology centre. One hundred eleven patient-episodes of fever and neutropenia were evaluated during the study period, 17 of which were associated with positive blood cultures, as follows:Staphylococcus epidermidis (n=6 episodes), Enterococcus faecium (n=2), Streptococcus sanguis (n=3), Streptococcus mitis (n=3), Staphylococcus aureus (n=1), Micrococcus spp. (n=1), and Stenotrophomonas maltophilia (n=1). Eubacterial polymerase chain reaction (PCR) detected bacterial DNA in nine of 11 blood culture-positive episodes for which a sample was available for PCR; the species identified by sequence analysis were identical to those derived from the conventional identification of the cultured isolates. Bacterial DNA was detected in 20 episodes (21 bacterial sequences) associated with negative blood cultures, 18 of which occurred in patients who were receiving antibiotics at the time of sample collection. The species presumptively identified by partial 16S rRNA gene sequencing were as follows:Pseudomonas spp. (n=6 episodes), Acinetobacter spp. (n=5), Escherichia spp. (n=3);Moraxella spp. (n=3);Staphylococcus spp. (n=2);Neisseria spp. (n=1); and Bacillus spp. (n=1). The results of this study suggest that molecular techniques can augment cultural methods in the diagnosis of bacteraemia in patients who have been treated with antibiotics.