Introduction One of the main goals of ethnomycological studies is to understand the role of wild edible mushrooms (WEM) in diverse cultures. To that end, the local knowledge of WEM and their cultural importance are evaluated and compared using qualitative and quantitative methods. However, few studies have attempted to discern such aspects about non-used mushrooms, since they are considered as a residual category without cultural relevance. Therefore, this paper analyses the traditional knowledge of non-edible mushrooms to understand their cultural role and break it down in its components. Methods This study was carried out in two communities from La Malintzi National Park, Tlaxcala, Mexico. Mushroom species indicated as non-edible were collected during 13 ethnomycological expeditions and 7 requests. To get an insight of local knowledge, we used ethnographic techniques, 91 free listings and 81 semi-structured interviews.Results In total, we collected 178 specimens of wild mushrooms recognized as non-edible by locals, which corresponded to 103 species belonging to 45 genera. The people who participated in the study had a vast and deep understanding of non-edible mushrooms. To avoid intoxications, all non-edible mushrooms were included in the general category of “poisonous mushrooms”. The knowledge and the nomenclature of non-edible mushrooms, as well as the ability to identify them, all emanate from the Mesoamerican duality conceptualization of the universe. Therefore, non-edible species are conceived as a cosmogonic counterpart ("twins") of the edible species to which they resemble. We obtained 101 specific recognition criteria, useful only when comparing species pairs: edible vs non-edible. The most culturally important non-edible groups were differentiated by clear and precise characteristics, which were reflected in the nomenclature and allowed their classification into specific ethnotaxa. Conclusions We found that non-used resources can be the object of a deep traditional knowledge and have a vast cultural importance. In the case of wild non-edible mushrooms in particular: the species are named, they are the subject of a vast traditional knowledge which is based on the edible/non-edible duality, this knowledge is widespread but has limited consensus, there is little lexical retention, and this knowledge is vital to avoid fatal intoxications. In consequence, deadly species and species that share similarities with the most important edible mushrooms have a high cultural importance.