Clostridium difficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MTR), vancomycin (VAN), clindamycin (CLI) and linezolid (LZD) on growth, toxin gene transcription and toxin production in C. difficile was investigated. Four C. difficile strains were grown with and without sub-MIC concentrations of MTR, VAN, CLI and LZD (0.5× MIC) and growth was measured by colony counts. Toxin production was detected using ELISA (for toxin A) and a cytotoxicity assay (for toxin B) in culture supernatants and also in sonicated cells. Real-time PCR was used to measure transcription of the toxin A and B genes. The aim of this work was to combine analysis of toxin A and B production by ELISA or cell culture assay with transcriptomic analysis. The four strains showed similar growth and different levels of toxin production in the absence of antibiotics. An antibiotic-free control showed toxin production at a late stage when the plateau phase of bacterial growth was reached, whereas antibiotic-exposed strains showed earlier toxin production. All of the antibiotics used except CLI increased the transcription rate of toxin genes. The findings of this study show that sub-MIC concentrations of antibiotics can cause changes in gene transcription of the major virulence factors of C. difficile. This study describes a new method for transcriptomic analysis of toxin genes in C. difficile.