Yearly the mushroom industry generates several million tons of spent mushroom substrate (SMS), a mixture of composted soil and fungal mycelium, left after the mushroom harvest. Although containing valuable ingredients like carbohydrates, lignin, and diverse enzymes, the substrate is unutilized and causes immense disposal costs. In order to valorize SMS, it is essential to fractionate the complex mixture into its valuable components, which is a challenge for current biorefineries and has only been partly achieved. We have developed a novel biorefinery strategy in order to separate carbohydrates and soluble lignin from SMS. Therefore, SMS was subjected to two different extraction methods in order to break the insoluble biopolymer residues: A, a thermochemical treatment (water/basic or acidic catalyst) yielding a carbohydrate-enriched liquid fraction; B, an organosolv extraction (with ethanol/water) solubilizing mainly lignin. The carbohydrate fraction possesses surface-active properties and was investigated as a potential biobased surfactant. The soluble lignin fraction was used for the formation of nanocarriers via an inverse miniemulsion polymerization. The lignin-based nanocarriers were biodegradable by laccases, which renders them of high interest for drug delivery systems for advanced plant protection. This novel biorefinery is a powerful strategy for the upcycling of SMS into various high-value products.