Background: Eastern Himalayas have rich biodiversity but are threatened by anthropogenic activities and periodic natural calamities. The present investigation focused on the cryptogamic group of macrofungi in the Darjeeling Himalayas which are extremely diverse and ecologically significant, but have received very little attention from a conservation perspective. Material and Methods: Investigations were conducted during June-September of 2013-2014 in the Darjeeling district of West Bengal, India, wherein, local macrofungi were sampled in mixed to monoculture forests using 50 random transects 100×2 m. Fruit bodies were studied, collected, identified in the laboratory and deposited at Calcutta University Herbarium. Sampling units were georeferenced and physico-climatic factors were derived from the WORLDCLIM database. Results: The macrofungi belonged to 98 species representing 72 genera and 47 families; 58.16% were saprotrophs, 17.34% were ectomycorrhizal and 10.2% were parasitic. The most species-rich families were Russulaceae with 9 Russula species followed by Marasmiaceae with 4 Marasmius species and Coprinaceae with 3 Coprinus species. Most of the ectomycorrhizal macrofungi were hosted by natural stands of Quercus, Lithocarpus and Betula. The only species with zooparasitic capability were Cordyceps nutans and C. militaris. The relative abundance of only 6 saprotrophs communicatively accounted for 52.84% of the total abundance. Conclusions: The findings showed that this region is rich in macrofungal diversity intricately linked to the functioning of the local ecosystem. However, a high proportion of saprotrophs compared to ectomycorrhizal species suggests disturbance and degradation of regional forests. Mycofloristically important localities like Lolaygaon, Sonada, and Lameydhura in the Darjeeling district should be prioritized for further studies and future conservation action. © 2016 Pradhan et al.