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Fungi are natural decomposers that degrade organic substrates for growth. On Earth, fungi grow and produce mushrooms on various natural substrates, often with little to no added nutrient supplements. Existing waste substrates found on board the International Space Station (ISS) such as inedible biomass from plants, clothing, and plastic wastes from prepackaged foods could be repurposed for food production and advance the capacity for more sustainable long-duration space missions. The sporeless oyster mushroom (Pleurotus ostreatus) strain SPX was grown on seven substrates in varied combination recipes to investigate how ISS waste streams could be used to cultivate mushrooms. In addition, food safety analyses were performed to assess the feasibility of mushroom cultivation as a low-risk food option. Results show that waste streams of cotton t-shirts and inedible biomass from plants are potential substrates that could support mushroom cultivation on board the ISS. By using materials that are already available on the station, the upmass needed to support such efforts is reduced and waste products can be recycled to potentially yield more food. This investigation was intended to identify the feasibility of incorporating mushrooms as a potential space crop without the requirement of a large upmass of substrates being brought to the ISS.