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In recent years, fungi, particularly lignicolous fungi, have been re-considered as a source for biotechnological and industrial applications. Lignicolous basidiomycetes are the most effective at degrading wood, particularly cellulose, hemicelluloses and lignin, which are among the most resistant biopolymers. This study aims to constitute a research collection of lignicolous fungal strains that are useful for further studies and applications in different production fields. The basidiomata used to isolate the strains in a pure culture were, firstly, identified through macroscopic and microscopic characteristics integrated with ecological data. To obtain pure cultures of dikaryotic mycelia, 96 different strains of Agaricomycetes belonging to 76 different species and related to 51 genera (18 families and 5 orders) were isolated using a malt extract agar (MEA) medium enriched with hydrogen peroxide. The identity of the isolated strains was then confirmed by molecular analysis through the sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster. All the strains are currently conserved using different methods, and their vitality is periodically tested.