In this study, mixed cultures of brown-rot fungus Gloeophyllum trabeum and filamentous fungus Trichoderma viride in decolorizing Methylene Blue (MB) has been investigated. T. viride culture was mixed with G. trabeum culture and then MB was added into the media to achieve final concentration 95.24 mg/L. The results showed single culture of G. trabeum and T. viride can decolorize MB only 31.50% and 53.89%, respectively. While decolorization of MB by mixed cultures of G. trabeum and T. viride reached to 74.72% in a liquid medium of potato dextrose broth (PDB) for 7 days. The results showed mixed cultures had the highest ability to decolorize MB among those variations. Based on the LC- TOF/MS analysis, the peak of MB in mixed cultures treatment had a lower intensity compared to the peak of MB in abiotic control. The metabolites from MB biodecolorization by G. trabeum were C8H10N2SO5, C6H8N2SO3, C16H20N3SO, C6H7NSO3, and C12H10N3S, while the metabolites from MB biodecolorization of T. viride were C16H23N3O3S, C22H26NO2S, and C31H48N3S. On the other hand, the metabolites from the MB biodecolorization by mixed cultures were C16H23N3O3S, C31H48N3S, C31H47N2OS and C17H20N2S. This study indicated that mixed cultures of brown-rot fungus G. trabeum and filamentous fungus T. viride was effective to decolorize MB dye.