The Andes range in Ecuador presents high biodiversity and characteristic altitudinal gradients, which are frequently threatened by deforestation and farming. In particular, forest have developed in the high inter-Andean alley on volcanic soils forming a unique ecoregion. Little is known on the fungal biodiversity of soil in such high Andean gallery forest submitted to strong degradation pressures. Therefore, in this study we evaluated wether the soil mycobiome was associated with altitudinal gradients during the dry season. Three representative locations were selected based on altitude: A (3,309 meters above the sea level, masl), B (3,809 masl) and C (4,409 masl). High performance sequencing (NGS) of the ITS region of ribosomal DNA genes with Illumina technology was used to explore the fungal taxonomic composition in the soil samples. Our results showed changes in the structure of fungal communities in the different locations, related to the relative abundance of Amplicon Sequence Variants (ASV). Higher fungal diversity was related with the altitudinal gradient with average taxa ranging from 675, 626 and 556 ASVs, respectively from location A to C. The results highlight the complexity and diversity of fungal communities in high Andean forest and the need to protect these unique mycobiomes. The findings in this ecosystem of Ecuador will improve our understanding of distribution, diversity, ecology, and biological perspectives for the restoration of terrestrial microbiomes.