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Late wilt disease (LWD) is a destructive vascular disease of maize (<i>Zea mays</i> L.) caused by the fungus <i>Magnaporthiopsis maydis.</i> Restricting the disease, which is a significant threat to commercial production in Israel, Egypt, Spain, India, and other countries, is an urgent need. In the past three years, we scanned nine <i>Trichoderma</i> spp. isolates as biological control candidates against <i>M. maydis</i>. Three of these isolates showed promising results. In vitro assays, seedlings pathogenicity trials, and field experiments all support the bio-control potential of these isolates (or their secretions). Here, a dedicated effort led to the isolation and identification of an active ingredient in the growth medium of <i>Trichoderma asperellum</i> (P1) with antifungal activity against <i>M. maydis</i>. This <i>Trichoderma</i> species is an endophyte isolated from LWD-susceptible maize seeds. From the chloroform extract of this fungal medium, we isolated a powerful (approx. 400 mg/L) active ingredient capable of fully inhibiting <i>M. maydis</i> growth. Additional purification using liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) separation steps enabled identifying the active ingredient as 6-Pentyl-α-pyrone. This compound is a potential fungicide with high efficiency against the LWD causal agent.