Mushroom waste can account for up to 50% of the total mushroom mass. Spent mushroom substrate, misshapen mushrooms, and mushroom stems are examples of mushroom byproducts. In ancient cultures, fungi were prized for their medicinal properties. Aqueous extracts containing high levels of β-glucans as functional components capable of providing prebiotic polysaccharides and improved texture to foods have been widely used and new methods have been tested to improve extraction yields. Similarly, the addition of insoluble polysaccharides controls the glycemic index, counteracting the effects of increasingly high-calorie diets. Numerous studies support these benefits in vitro, but evidence in vivo is scarce. The aim of this project is to obtain potential extracts of mushroom by-products as organic sources of antioxidants, antimicrobials, and prebiotics, creating new avenues for food product development. Reusing these by-products could encourage sustainability and assist the mushroom sector in implementing zero-waste and circular economy methods.