Microfungal applications are increasing daily in the medical science. Several species of Trichoderma are widely used in agricultural fields as biological control and plant growth promoting agents. The application of Trichoderma asperellum as an entomopathogenic fungus against the Anopheles mosquito, a vector of malaria, is a novel control approach. Controlling malaria with eco-friendly management practices is an urgent need. We isolated three T. asperellum from different natural sources using serial dilution and mosquito baiting techniques. The fungi were identified on the basis of phenotypical and molecular characteristics. The fungi were grown in different natural media to examine spore production ability and the fungal spore suspensions were applied to the anopheline larvae to determine their larvicidal activity in vitro. We investigated the efficacy of crude ME (methanolic extract) and different methanolic fractions (MFs) of the fungal extracts against anopheline larvae. Methanolic Fraction 8 (MF8) exhibited the strongest larvicidal activity. A GC-MS analysis of MF8 and a Chemolibrary search were performed to identify the active agents in the fungal extracts. Among the three isolates of T. asperellum, the TaspSKGN2 isolate showed the lowest LD50 (2.68 × 107 conidia/mL) and LT50 values (12.33 h). The crude ME exhibited LD50 values of 0.073 mg/mL and LT50 values of 11.33 h. MF8 showed LD50 values of 0.059 mg/mL and LT50 values of 8.57 h. In GC-MS study of MF8, 49 compounds were found. Among these, seven compounds (2,3-di hydro thiopene, p-cymene, alpha-pinene, hexadecanoic acid, 8-methyl quinoline, (Z,Z)-9,12-octa decadienoic acid, methyl ester, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-Pyran-4-one-) with high abundance were found to have insecticidal efficacy by a literature survey. We detected a reduction in the phenoloxidase content inside the cuticle and hemolymph of the anopheline larvae after a few hours of interaction with ME (0.073 mg/mL). Thus Trichoderma asperellum has new applications for the control of Anopheles spp. malaria vectors.