We investigated the performance of cefotaxime for the detection of extended-spectrum β-lactamase (ESBL) or plasmid-mediated AmpC β-lactamase (pAmpC) and the clinical characteristics of cefotaxime-non-susceptible Escherichia coli or Klebsiella pneumoniae (CTXNS-EK) bacteraemia. All of the consecutive bloodstream isolates between 2005 and 2010 in a Japanese university hospital were characterised using polymerase chain reaction (PCR). Risk factors and outcomes of CTXNS-EK were analysed by multivariate logistic regression analysis. We identified 58 CTXNS-EK (15.6%) from 249 E. coli and 122 K. pneumoniae. Cefotaxime with a minimum inhibitory concentration (MIC) of >1 μg/mL had a sensitivity of 98.3% and a specificity of 99.7% for the detection of ESBL or pAmpC. CTXNS-EK had increased from 4.5% in 2005 to 23% in 2009. Risk factors for CTXNS-EK were previous isolation of multidrug-resistant bacteria, use of oxyimino-cephalosporins or fluoroquinolones, and high Sequential Organ Failure Assessment (SOFA) score. Patients with CTXNS-EK bacteraemia less frequently received appropriate empirical therapy than patients with cefotaxime-susceptible EK bacteraemia (81% vs. 97%, p<0.001) and died within 30 days (21% vs. 5%, p=0.001). Using the current breakpoints of the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), cefotaxime alone can identify ESBL or pAmpC producers. CTXNS-EK is an important and increasingly prevalent bacteraemia pathogen.