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Objective The toxin-producing Klebsiella oxytoca causes antibiotic-associated haemorrhagic colitis (AAHC). The disease-relevant cytotoxins tilivalline and tilimycine produced by certain K. oxytoca isolates are encoded by the non-ribosomal peptide synthetase genes A (npsA) and B (npsB). In this study, the new LightMix® Modular kit for detection of relevant K. oxytoca sensu lato (s.l.) toxin genes was evaluated. Methods DNA was extracted on the automated EMAG® platform. Amplification was done on the Light Cycler® 480 II instrument. A total of 130 residual faecal specimens that had been collected from patients with antibiotic-associated diarrhoea were studied to determine the clinical sensitivity and specificity. Toxigenic culture served as reference method. Results With the new kit, the limit of detection was 15 CFU/mL for all targets. For the K. oxytoca s.l. specific pehX target, 65 of 130 clinical specimens were positive while toxin specific targets (npsA/npsB) were positive in 47 of 130. The npsA/npsB PCR targets showed a clinical sensitivity of 100% (95% CI of 80.5% to 100%) and a specificity of 73.5% (95% CI of 64.3% to 81.3%) with a positive predictive value of 16.5% (95% CI of 12.7% to 21.2%) and a negative predictive value of 100%. Conclusion Compared with culture, additional K. oxytoca s.l.-positive clinical specimens were detected with real-time PCR. The specificity of the toxin targets appears moderate due to the inferior sensitivity of the culture-based reference method. Since the developed assay is highly sensitive, it may be used as first-line method to improve AAHC diagnosis.