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To collect and directionally explore siderophore-producing microorganisms, the siderophore-producing microorganisms in the native forest soil of the Ailao Mountain were screened, isolated, and identified using the pure culture method. Combined with the high-throughput sequencing results of soil internal transcribed spacer (ITS) amplicons, the position of active strains in the community structure was anchored. The relationship between active strains and environmental factors was analyzed further. The results showed that all of the active siderophore-producing fungal strains screened and isolated from the Ailao Mountain valley and the original forest soils through pure culture were Ascomycetes, which belonged to Aspergillus, Penicillium, Metarhizium, Trichoderma, Talaromyces, and Beauveria. These microorganisms were mainly isolated from high altitude virgin forest areas, in which most of the soil samples had high organic matter content, high water content, low pH value, and low ferrous content. Combined with the ITS high-throughput sequencing results, the above active strains all had the corresponding operational taxonomic units (OTUs). The results of the OTUs-environmental factor analysis showed that the Penicillium strains were significantly positively correlated with organic matter content; Aspergillus strains were significantly positively correlated with pH value but had a significant negative correlation with altitude and soil water content; Trichoderma and Talaromyces strains had not significantly correlated with all environmental factors; the distribution of Beauveria and Metarhizium had certain positive correlation with pH value. Thus, it is proposed that the primitive forest environment has a selective impact on the microorganisms. The siderophore-producing Ascomycetes from the primitive forest are important functional resources worthy of further exploration and may play an important role in the circulation of soil minerals and organic matter formation. [ABSTRACT FROM AUTHOR]