Abstract There has been an increase in the occurrence of fungal diseases in Ilex paraguariensis A. St.-Hill. (yerba mate) resulting from their domestication. Seed health tests may indicate pathogens prior to sowing, enabling their control. Our study sought to compare the efficacy of fungi detection methods on yerba mate seeds. Seeds from four lots were subjected to the following detection methods: BDA, V8 and filter paper; as well as the performance of asepsis with 1% sodium hypochlorite and 70% alcohol. The fungi found in the lots were: Penicillium spp., Cladosporium spp., Aureobasidium melanogenum, Aspergillus spp., Pestalotia spp., Nigrospora spp., Trichoderma spp., Fusarium graminearum, F. oxysporum and F. guttiforme. The asepsis in the seeds reduced the occurrence of contaminating organisms. There were differences between the evaluated lots regarding fungal incidence. In general, the BDA and V8 culture media showed better results in fungal detection.