Macrofungi are classified in the phylum Ascomycota and Basidiomycota and they are very important from an ecological and economic point of view. Most studies of fungi in Colombia have been carried out mainly in the Andean Region, especially in the Departments of Antioquia, Valle del Cauca and Cundinamarca. However, other Departments in the Andean Region, like Tolima, located in the Cordillera Central, are well documented for plants (4,797 species) and animals (2,983 species), but very poorly documented in terms of knowledge of fungal diversity. This study provides a compiled and annotated checklist of all known macrofungi in the Department of Tolima, based on published literature and on the identification of new specimens collected from five localities of the Department. All records were updated taxonomically and we include detailed information on the localities in which they are distributed in the Department. The list includes 164 taxa distributed in 15 orders (Agaricales, Polyporales, Russulales, Boletales, Hymenochaetales, Xylariales, Auriculariales, Thelephorales, Cantharellales, Hypocreales, Pezizales, Gloeophyllales, Phallales, Tremellales, Dacrymycetales) and eighteen records in a doubtful taxa section. We present 26 new reports, 19 for Tolima and nine for Colombia. We also provide genetic and phylogenetic evidence of the occurrence of Gloeoporus telephoroides and Podoscypha venustula in Colombia. This checklist provides the basis for future studies on species diversity and taxonomy in Tolima, by identifying the least studied taxa and ecosystems and conservation priorities.