The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the morphotype responsible for transmission, infection, and persistence, and the outermost exosporium layer is likely to play a major role in spore-host interactions during recurrent infections, contributing to the persistence of the spore in the host. A recent study (M. Pizarro-Guajardo, P. Calderon-Romero, P. Castro-Cordova, P. Mora-Uribe, and D. Paredes-Sabja, Appl Environ Microbiol 82:2202-2209, 2016, http://dx.doi.org/10.1128/AEM.03410-15) demonstrated by transmission electron microscopy the presence of two ultrastructural morphotypes of the exosporium layer in spores formed from the same sporulating culture. However, whether these distinct morphotypes appeared due to purification techniques and whether they appeared during biofilm development remain unclear. In this communication, we demonstrate through transmission electron microscopy that these two exosporium morphotypes are formed under sporulation conditions and are also present in spores formed during biofilm development. In summary, this work provides definitive evidence that in a population of sporulating cells, spores with a thick outermost exosporium layer and spores with a thin outermost exosporium layer are formed.