In this study, the potential effects of bacteria on the efficacy of frequently used chemotherapies was examined. Bacteria and cancer cell lines were examined in vitro and in vivo for changes in the efficacy of cancer cell killing mediated by chemotherapeutic agents. Of 30 drugs examined in vitro, the efficacy of 10 was found to be significantly inhibited by certain bacteria, while the same bacteria improved the efficacy of six others. HPLC and mass spectrometry analyses of sample drugs (gemcitabine, fludarabine, cladribine, CB1954) demonstrated modification of drug chemical structure. The chemoresistance or increased cytotoxicity observed in vitro with sample drugs (gemcitabine and CB1954) was replicated in in vivo murine subcutaneous tumour models. These findings suggest that bacterial presence in the body due to systemic or local infection may influence tumour responses or off-target toxicity during chemotherapy.