Shigatoxigenic Escherichia coli (STEC) such as E. coli O157 are significant human pathogens, capable of producing severe, systemic disease outcomes. The more serious symptoms associated with STEC infection are primarily the result of Shiga toxin (Stx) production, directed by converting Stx bacteriophages. During phage-mediated replication and host cell lysis, the toxins are released en masse from the bacterial cells, and the severity of disease is linked inexorably to toxin load. It is common for a single bacterial host to harbour more than one heterogeneous Stx prophage, and it has also been recently proven that multiple isogenic prophage copies can exist in a single cell, contrary to the lambda immunity model. It is possible that in these multiple lysogens there is an increased potential for production of Stx. This study investigated the expression profiles of single and double isogenic lysogens of Stx phage 24(B) using quantitative PCR to examine transcription levels, and a reporter gene construct as a proxy for the translation levels of stx transcripts. Toxin gene expression in double lysogens was in excess of the single lysogen counterpart, both in the prophage state and after induction of the lytic life cycle. In addition, double lysogens were found to be more sensitive to an increased induction stimulus than single lysogens, suggesting that maintenance of a stable prophage is less likely when multiple phage genome copies are present. Overall, these data demonstrate that the phenomenon of multiple lysogeny in STEC has the potential to impact upon disease pathology through increased toxin load.