Amidochelocardin is an atypical tetracycline which was previously shown to target the bacterial cell membrane although its detailed mode-of-action is still unknown. To gain insight into Clostridioides difficile’s response to membrane targeting antibiotics and to further study the mode-of-action of Amidochelocardin, the stress response of C. difficile 630 to sublethal doses of amidochelocardin were studied on the proteome level. The results and results from downstream experiments, such as quantification of the membrane potential, cellular localization assays and transmission electron microscopy analysis, suggest that amidochelocardin kills bacteria such as C. difficile by dissipating their membrane potential. Furthermore, the data suggest that C. difficile responds to dissipation of the membrane potential by induction of biosynthesis of an hitherto unknown aromatic compound.