The Cape Horn Biosphere Reserve, Chile, has been identified as a hotspot of bryophyte diversity and it has been suggested to be the same for lichens. However, in contrast to the extensive bryophyte studies, only preliminary lichen inventories had been conducted in this reserve. We conducted the first intensive study on the diversity of lichens on Navarino Island during the southern summers of 2005 and 2008. We explored the main habitat types of the island, including coastal areas, evergreen and deciduous forests, Magellanic tundra, and high Andean (“alpine”) habitats on the mountain summits. The following substrates on which lichens grow were considered: bark, wood (incl. logs, stumps), soil, mosses, and rocks. We recorded a total of 416 taxa, although some of them not identified to species level. A main result is the finding of two species that are proposed as new: The lichen Candelariella magellanica, and the saprobic fungus Sclerococcum nothofagi that grows on the bark of trees of the genus Nothofagus. In addition, one species of lichenicolous fungus is recorded for the first time on Navarino Island: Tremella haematommatis. These results provide additional evidence about the great diversity of lichens that are conserved in the Cape Horn Biosphere Reserve.