Studies on primary succession and biological surveys in Surtsey have described the presence of different organisms including plants, soil fauna and fungi. However, since the last fungal census of 2008, based on visual observations, no further studies on fungal communities on the island have been published. In the present study, we aimed to expand the knowledge of the fungal communities of Surtsey using Internal Transcribed Spacer (ITS2) metabarcode sequencing to survey soil samples from 23 permanent plots, within and outside a gull colony. Additionally, we related fungal community composition to soil and ecosystem environmental variables that have been analyzed from these plots previously. We report at least 18 new genera and 44 new species for Iceland and 56 new species for Surtsey in this metabarcode survey, likely representing a combination of established organisms as well as those that may be present as dormant propagule bank. Our data indicate that fungal communities differ in areas with and without sea gulls as well as those that differ in soil substrate. Further, the community composition correlates with the number of gull nests, vegetation cover, ecosystem respiration, total N and exchangeable P. This survey provides new insights on the fungal community dynamics in relation to other biotic and abiotic factors. These findings complement what available data on soil biosphere on Surtsey and improve our understanding of primary succession.