| Summary: | Fungal endophytes can protect plants against herbivory and be used to control leaf-cutting ants. In this study, we aimed to evaluate the potential of endophytic colonization of <i>Eucalyptus urophylla</i> by three filamentous fungal species and their influence on the plant development and foraging behavior of <i>Atta sexdens</i>. The study design was completely randomized and comprised a factorial scheme of 4 × 3, three antagonistic fungal species (<i>Escovopsis</i> sp., <i>Metarhizium anisopliae</i>, and <i>Trichoderma strigosellum</i>) of the leaf-cutting ant, and one control and three inoculation methods (conidial suspension via foliar spray [FS] and soil drench [SD] inoculation, and seedlings inoculated with mycelium [SWM]). The SWM method allowed <i>T. strigosellum</i> to colonize all plant organs, and these plants exhibited higher height, leaf number, shoot dry mass, and total dry mass than the ones subjected to the other inoculation methods. The SWM method increased the plant height than the control plants and those inoculated with <i>Escovopsis</i> sp. and <i>M. anisopliae</i>. <i>Trichoderma strigosellum</i>, previously isolated from soil, colonized <i>E. urophylla</i> plants and positively influenced their development, as demonstrated by the SWM method. <i>Trichoderma strigosellum</i> promoted the increase in <i>E. urophylla</i> height compared with when the FS and SD methods were used (by 19.62% and 18.52%, respectively). Our results reveal that <i>A. sexdens</i> workers preferentially began cutting the leaves from plants not previously colonized by <i>T. strigosellum</i>. This behavior can be explained by modifications in the phenotypic traits of the eucalyptus leaves.
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