Rot disease, caused by the fungal pathogen Peniophora salaccae SKRU002, affects the quality of snake fruit production. In the pursuit of sustainable disease management, biocontrol using Trichoderma asperelloides SKRU-01 offers a promising solution. This study evaluated the antagonistic potential of T. asperelloides SKRU-01 against P. salaccae SKRU002 in both in vitro assays and snake fruit trials, while also assessing its impact on fruit quality. In vitro dual culture assays revealed that T. asperelloides SKRU-01 inhibited P. salaccae SKRU002 growth by 62.5 % over 10 days through efficient nutrient colonization. Microscopic analysis confirmed that T. asperelloides SKRU-01 hyphae penetrated and wrapped around P. salaccae SKRU002, causing cytoplasmic lysis. Additionally, T. asperelloides SKRU-01 culture filtrates (20 % v/v) completely inhibited P. salaccae SKRU002 growth in both solid and liquid media. LC-QTOF/MS analysis identified 31 secondary metabolites, including toyocamycin and antimycin A1. In snake fruit trials, T. asperelloides SKRU-01 culture filtrates provided 100 % protection against disease incidence (DI) and severity (DS), comparable to Mancozeb®. The application of T. asperelloides SKRU-01 spores (107 spores/mL) reduced DI and DS to 0 % within 1-3 days post-pathogen inoculation, demonstrating both protective and curative effects. Furthermore, while P. salaccae SKRU002 significantly affected fruit quality-causing weight loss, color changes, and reductions in total soluble solids, phenolic content, titratable acidity, and antioxidant activity-the simultaneous application of T. asperelloides SKRU-01 mitigated these effects without compromising fruit quality. These findings indicate the antagonistic activity of T. asperelloides SKRU-01 and its metabolites against P. salaccae SKRU002, suggesting their potential as biofungicidal agents for managing rot disease in snake fruit.