| Summary: | Flower blight caused by <i>Neopestalotiopsis clavispora</i> is an emerging disease of flamingo flower (<i>Anthurium andraeanum</i> Lind.) that negatively impacts flower production. The use of rhizosphere fungi as biocontrol agents is an alternative way to control this disease instead of using synthetic fungicides. This research aimed to screen the potential of rhizosphere fungi, <i>Trichoderma</i> spp., with diverse antifungal abilities to control <i>N. clavispora</i> and to reduce flower blight in flamingo flowers. A total of ten isolates were tested against <i>N. clavispora</i> by dual culture assay, and T1-02 was found to be the most effective isolate against <i>N. clavispora</i>, with inhibition of 78.21%. Morphology and molecular phylogeny of multiple DNA sequences of the genes, the internal transcribed spacer (ITS), translation elongation factor 1-α (<i>tef1-α</i>), and RNA polymerase 2 (<i>rpb2</i>) identified isolate T1-02 as <i>Trichoderma virens</i>. Sealed plate method revealed <i>T. virens</i> T1-02 produced volatile antifungal compounds (VOCs) against <i>N. clavispora</i>, with inhibition of 51.28%. Solid-phase microextraction (SPME) was applied to trap volatiles, and GC/MS profiling showed VOCs emitted from <i>T. virens</i> T1-02 contained a sesquiterpene antifungal compound—germacrene D. The pre-colonized plate method showed that <i>T. virens</i> T1-02 aggressively colonized in tested plates with inhibition of 100% against <i>N. clavispora</i>, and microscopy revealed direct parasitism onto fungal hyphae. Furthermore, the application of <i>T. virens</i> T1-02 spore suspension reduced the disease severity index (DSI) of flower blight in flamingo flowers. Based on the results from this study, <i>T. virens</i> T1-02 displays multiple antagonistic mechanisms and has the potential ability to control flower blight of flamingo flowers caused by <i>N. clavispora</i>.
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