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The dynamic of soil-borne disease is closely related to the rhizosphere microbial communities. Maize–soybean relay strip intercropping has been shown to significantly control the type of soybean root rot that tends to occur in monoculture. However, it is still unknown whether the rhizosphere microbial community participates in the regulation of intercropped soybean root rot. In this study, rhizosphere <i>Fusarium</i> and <i>Trichoderma</i> communities were compared in either healthy or root-rotted rhizosphere soil from monocultured and intercropped soybean, and our results showed the abundance of rhizosphere <i>Fusarium</i> in intercropping was remarkably different from monoculture. Of four species identified, <i>F. oxysporum</i> was the most aggressive and more frequently isolated in diseased soil of monoculture. In contrast, <i>Trichoderma</i> was largely accumulated in healthy rhizosphere soil of intercropping rather than monoculture. <i>T. harzianum</i> dramatically increased in the rhizosphere of intercropping, while <i>T. virens</i> and <i>T. afroharzianum</i> also exhibited distinct isolation frequency. For the antagonism test in vitro, <i>Trichoderma</i> strains had antagonistic effects on <i>F. oxysporum</i> with the percentage of mycelial inhibition ranging from 50.59–92.94%, and they displayed good mycoparasitic abilities against <i>F. oxysporum</i> through coiling around and entering into the hyphae, expanding along the cell–cell lumen and even dissolving cell walls of the target fungus. These results indicate maize–soybean relay strip intercropping significantly increases the density and composition proportion of beneficial <i>Trichoderma</i> to antagonize the pathogenic <i>Fusarium</i> species in rhizosphere, thus potentially contributing to the suppression of soybean root rot under the intercropping.