Managing plant diseases caused by phytopathogenic fungi, like anthracnose caused by Colletotrichum species, is a challenge. Different methods are being sought to identify compounds with antibiotic properties. The Trichoderma strains represent a source of novel molecules with antifungal properties, including Volatile Organic Compounds (VOCs), the production of which is influenced by the medium’s nutrient content. In this study, we assessed the VOCs produced in dual confrontation systems performed in two culture media by Trichoderma strains (T. atroviride IMI206040, T. asperellum T1 and T3, and Trichoderma sp. T2) against Colletotrichum acutatum. We analyze the VOCs profiles using gas chromatography coupled with mass spectrometry. The Luria Bertani medium (LB) stimulated VOCs production with antifungal properties in most systems. We determined the 2-pentyl furan, dimethyl disulfide, and α-phellandrene antifungal activity in vitro. The equimolar mixture of those VOCs (250 µM) generated 14% C. acutatum diametral growth inhibition. The infective ability and the disease severity caused by the mycelium exposed to the VOCs mixture significantly diminished on strawberry leaves. The application of those VOCs as biofumigants could contribute to managing anthracnose. The use of LB represents a feasible strategy to identify VOCs with antifungal properties even between Trichoderma strains belonging to the same species.