The results of a large-scale biodiversity inventory of lichens (including lichenicolous and allied fungi) in the Dare Regional Biodiversity Hotspot (DRBH) are presented. The DRBH is a region within the Mid-Atlantic Coastal Plain (MACP) of eastern North America that was recently delineated based on its unique and diverse lichen communities relative to other areas of the Atlantic Coast. Drawing on 4,952 newly generated voucher specimens from 49 sites, patterns of biodiversity and biogeography are presented and discussed within the context of both the DRBH and the broader MACP. Relationships between natural communities, vegetation, and lichen communities are discussed, as are threats to the lichen biota. A series of conservation actions are presented together with avenues for future study. In addition, supplementary resources are provided in the form of: (a) a checklist of DRBH lichens, lichenicolous fungi, and allied fungi; (b) keys to DRBH lichens and lichenicolous and allied fungi; and (c) formal descriptions of the following species new to science that were discovered during the inventory: Albemarlea pamlicoensis gen. et. sp. nov., Arthonia agelastica sp. nov. (on Lecanora louisianae B. de Lesd.), Arthonia hodgesii sp. nov. (on Graphis lineola), Arthonia stevensoniana sp. nov. (on Haematomma accolens), Lichenochora haematommatum sp. nov. (on Haematomma persoonii), Megalaria alligatorensis sp. nov., Minutoexcipula miniatoexcipula sp. nov. (on Pertusaria epixantha), Trichosphaerella buckii sp. nov. (on Punctelia rudecta).