Aims The objective of this study was to determine the effects of Ca‐dipicolinic acid (CaDPA), cortex‐lytic enzymes (CLEs), the inner membrane (IM) CaDPA channel and coat on spore killing by dodecylamine. Methods and Results Bacillus subtilis spores, wild‐type, CaDPA‐less due to the absence of DPA synthase or the IM CaDPA channel, or lacking CLEs, were dodecylamine‐treated and spore viability and vital staining were all determined. Dodecylamine killed intact wild‐type and CaDPA‐less B. subtilis spores similarly, and also killed intact Clostridiodes difficile spores ± CaDPA, with up to 99% killing with 1 mol l−1 dodecylamine in 4 h at 45°C with spores at ~108 ml−1. Dodecylamine killing of decoated wild type and CLE‐less B. subtilis spores was similar, but ~twofold faster than for intact spores, and much faster for decoated CaDPA‐less spores, with ≥99% killing in 5 min. Propidium iodide stained intact spores ± CaDPA minimally, decoated CaDPA‐replete spores or dodecylamine‐killed CLE‐less spores peripherally, and cores of decoated CaDPA‐less spores and dodecylamine‐killed intact spores with CLEs. The IM of some decoated CaDPA‐less spores was greatly reorganized. Conclusions Dodecylamine spore killing does not require CaDPA channels, CaDPA or CLEs. The lack of CaDPA in decoated spores allowed strong PI staining of the spore core, indicating loss of these spores IM permeability barrier. Significance and Impact of the Study This work gives new information on killing bacterial spores by dodecylamine, and how spore IM’s relative impermeability is maintained.