In this paper, an annotated checklist of macrofungi from Őrseg National Park, West Hungary, is provided. A total of 726 macrofungi taxa representing 214 genera, 84 classes and 2 phyla (Ascoand Basidiomycota) were revealed. Sixty-one macrofungi species were new to the mycobiota of Hungary. Sporocarps were collected three times (in May, August and September–October) between 2009 and 2010 in 35 (40 m × 40 m) forest stands with different tree species compositions. Preferred tree species compositions and substrata of registered macrofungi are also listed.