The realm of biosensor development has expanded significantly with the inclusion of natural bioactive compounds, offering innovative solutions for enhanced detection and measurement capabilities. Mushrooms, known for their rich and diverse bioactive compound profile, emerge as a valuable resource in this context. This review delves into the applications of mushroom-derived compounds, such as enzymes, polysaccharides, and antioxidants, in biosensor technology. Enzymes like laccase and tyrosinase, sourced from various mushroom species, have demonstrated exceptional catalytic properties, making them ideal for biosensors aimed at detecting a wide range of substances, including phenols, glucose, and other organic compounds. Polysaccharides, particularly β-glucans, contribute to the structural matrix of biosensors, enhancing their stability and functionality. Additionally, antioxidants derived from mushrooms offer protective benefits, improving the longevity and reliability of biosensors by mitigating oxidative stress. Recent advancements highlight successful integrations of these compounds in biosensors, showcasing improved performance metrics compared to traditional biosensor components. For instance, laccase-based biosensors have been effectively utilized in environmental monitoring for detecting pollutants, while β-glucan-enhanced biosensors have shown promise in medical diagnostics for glucose monitoring. The environmental and economic benefits of using mushroom-derived compounds are significant. Mushrooms are renewable, biodegradable, and can be cultivated on a large scale with minimal environmental impact. This sustainable approach not only reduces dependency on synthetic materials but also aligns with the growing demand for eco-friendly technologies. However, the integration of mushroom-derived compounds in biosensors is not without challenges. Issues such as the consistency of compound extraction, stability under various conditions, and integration with existing sensor technologies require further research and development. Future directions include optimizing extraction methods, exploring novel mushroom species for bioactive compounds, and developing hybrid biosensors that leverage the strengths of both natural and synthetic materials.