Changes in soil fungal community caused by land use have not been sufficiently studied in South-American Andosols, considered globally as important food production areas. This study analyzed 26 soil samples of Andosols collected from locations devoted to conservation, agriculture and mining activities in the southeastern region of Antioquia, Colombia, to establish differences between fungal communities as indicators of the degree of soil perturbation. The study developed a novel heminested PCR with primers SSUmCf Mix, ITS4 and fITS7 to assess Arbuscular Mycorrhizal Fungi detection in a Illumina MiSeq metabarcoding on nuclear ribosomal ITS2 region. A non-metric multidimensional scaling allowed exploring driver factors of fungal community changes, while fitted Dirichlet-multinomial models and PERMANOVA tests allowed identifying the correlations between alpha diversity indexes and community dissimilarities, as well as the significance of land use effects on fungal community composition. Furthermore, response ratios were determined to assess effect size by land use over relevant taxa. Results suggest a good coverage of fungal diversity with a detection of 10,529 high-quality ITS2 sequences belonged to phylum Glomeromycota. The analysis shows strong correlations of Shannon and Fisher indexes with dissimilarities on fungal communities among land uses (r=0.94), related to variations in temperature, air humidity and organic matter contents that lead to significant responses in abundances of relevant orders (such as Wallemiales and Trichosporonales). The study highlights the rich fungal biodiversity of the tropical Andosols, their specific sensitivities to environmental perturbation factors, and the useful range of a metabarcoding approach to characterize soil fungal communities.