Milk thistle (Silybum marianum (L.) Gaertn.) is one of the most important medicinal plants. The fungi infesting its seeds may negatively influence their germination and health-promoting properties. However, there is no standard method for S. marianum seed health testing. The aim of this study was to find a suitable method for the detection and identification of fungi in/on milk thistle seeds. The following tests were used: deep-freeze blotter test, blotter test with an addition of mannitol, blotter test with an addition of polyethylene glycol, agar test on potato-dextrose agar (PDA), agar test on PDA after seed disinfection, agar test on reduced PDA and an agar test on reduced PDA after seed disinfection. Seeds were incubated for 10 and 14 days. The most prevalent fungi were: Alternaria alternata, Botrytis cinerea, Cladosporium spp., Fusarium spp., Penicillium spp., Rhizopus nigricans and Verticillium spp. A deep-freeze blotter test followed by a blotter test with an addition of mannitol and a blotter test with an addition of polyethylene glycol performed for 14 days could be recommended for the further study of milk thistle seed health testing, as they favoured the growth of the most important fungi.