Wheat straw (Triticum aestivum) is one of the lignocellulosic materials largely available worldwide and could be potentially used for biofuel production. Aiming the cost-effective utilization of wheat straw in the sugar-based biorefineries, co-cultures of Trichoderma reesei and Monascus purpureus were used for the enzymatic hydrolysis of the wheat straw biomass. The enzymatic breakdown of the dual-fungi-treated wheat straw was chemically analyzed through different enzyme/compositional assays, and the structural modifications were studied through scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR). For hydrolytic enzyme assays, the co-culture treatments resulted in significantly higher values (carboxymethyl cellulase (212.3 U/ml; p = 0.0173*), total cellulase (202 U/ml; p < 0.0001****), and xylanase (96.7 U/ml; p < 0.0001****) when compared with the readings of pure cultures. This hydrolytic activity resulted in the enhanced breakdown of wheat straw exhibiting a significant loss of 45.2% in lignin, 19.18% in cellulase, and 21.84% in hemicellulose contents. Furthermore, SEM and FTIR analysis of the co-culture treatments verified the improved biodegradation of wheat straw. Accumulatively, these results suggest a better approach for the effective use of dual-fungi for the lignocellulosic biomass breakdown and may have applications in bioethanol biorefineries using wheat straw as a sugar feedstock.