Soil fungi play an indispensable role in the functioning of terrestrial habitats. Most landscape-scale studies of soil fungal diversity try to identify the fungal taxa present at a study site and define the relationships between their abundance and environmental factors. The specific spatial distribution of these fungi over the site, however, is not addressed. Our study’s main objective is to propose a novel approach to landscape-scale mapping of soil fungi distribution using next generation sequencing and geographic information system applications. Furthermore, to test the proposed approach and discuss its performance, we aimed to conduct a case study mapping the spatial distribution of soil fungi on the Wielka Żuława island. The case study was performed on the Wielka Żuława island in northern Poland, where soil samples were collected every 100 m in an even grid. The fungal taxa and their relative abundance in each sample were assessed using the Illumina platform. Using the data obtained for the sampled points, maps of soil fungi spatial distribution were generated using three common interpolators: inverted distance weighted (IDW), B-spline, and ordinary Kriging. The proposed approach succeeded in creating maps of fungal distribution on Wielka Żuława. The most abundant groups of soil fungi werePenicilliumon the genus level, Aspergillaceae on the family level, and ectomycorrhizal fungi on the trophic group level. Ordinary Kriging proved to be the most accurate at predicting relative abundance values for the groups of fungi significantly spatially autocorrelated at the sampled scale. For the groups of fungi not displaying spatial autocorrelation at the sampled scale, IDW provided the most accurate predictions of their relative abundance. Although less accurate at predicting exact relative abundance values, B-spline performed best in delineating the spatial patterns of soil fungi distribution. The proposed approach to landscape-scale mapping of soil fungi distribution could provide new insights into the ecology of soil fungi and terrestrial ecosystems in general. Producing maps of predicted fungal distribution in landscape-scale soil fungi diversity studies would also facilitate the reusability and replicability of the results. Outside the area of research, mapping the distribution of soil fungi could prove helpful in areas such as agriculture and forestry, nature conservation, and urban planning.