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In semi-arid Mediterranean environments, fungal activity is fundamental for buffering biotic and abiotic stress to the plant and for sustaining a vegetation cover. Despite the important role that fungi play in habitats stability, mycological data from Mediterranean ecosystems are scarce and fragmentary. We investigated fungal diversity in several areas characterized by Mediterranean maquis, from continental Italy, Sicily, and Greece in order to contribute to the analysis of distribution, ecology, and diversity of macrofungi in evergreen sclerophyllous shrublands at different elevation and distance from the seashore across the Mediterranean Basin. Several fungal taxa that are remarkable due to their ecology, rarity, and limited geographical distribution were recovered. Among them, the ectomycorrhizal species Amanita dunensis and A. valens were recorded for the first time in Italy. The wood-inhabiting macrofungi, Aleurodiscus dextrinoideocerussatus, Peniophorella tsugae, Perenniporia meridionalis, Phanerochaete martelliana, Vararia ochroleuca, and six Peniophora species were reported for the first time in Greece. Some species, such as Gloeodontia columbiensis, Ceriporia aurantiocarnescens, Peniophora pithya, and P. tamaricicola were collected on new substrates. For the two rare basidiomycetes Amanita eliae and Battarrea phalloides, molecular identification and phylogenetic analysis gave valuable information on their relationship with similar taxa.