The present work describes the soil mycobiota in the upper AndeanColombian agro-environments using a meta-barcoding approach on fungal ITS1 region. Colombia is a country for which one of the highest biodiversity rates is reported, even if mycological data are scarce anddiscontinuous. The region sampled in this study belongs to the upper Andean zone and has a notable ecological and economical value:originally covered by forests, in the last 50 years human activities have turned it intograsslands partly devoted to subsistence agriculture. Recently, economically relevant cultureshave been introduced, such as fine varieties of fruit trees.The present meta-barcoding analysis wants to deepen the knowledge of the microbiological condition ofthis land, to guide future choices aimed at its sustainable management. The analysis revealed ca. 150 described species of fungi differently distributed inorchards and non-cultivated areas. The bootstrap-based clustering analysis evidences a clearresponse of fungal community to different use of soil in the agro-environment.