Astronauts on the International Space Station still depend on resources brought by cost-intensive resupply missions. To enhance their independence from Earth and prepare for long-term crewed missions to the Moon or Mars, there is an urgent need to develop self-sustained habitats that rely on in situ utilization of resources, thus reducing mass, space, and environmental constraints. One promising approach involves growing filamentous fungi on locally available nutrients and utilizing the fungi’s vast bioprocessing abilities to answer the crew’s needs in situ, e.g., through production of antibiotics, crop bioremediation, or biomining of lunar and Martian regolith. However, given the limited resources that can be transported for long-term space missions, the utilization of traditional culture media to support such production systems is impractical. Thus, in this work we developed an alternative culture medium using components present in space waste resources for growth of the fungus Penicillium simplicissimum, a commonly used model species in biotechnological applications. We identified 10 promising waste components and used Design of Experiments screening and optimization strategies to test 94 media formulations, including 32 runs in the screening step and 62 in the optimization step. Validation experiments demonstrated that the optimized space waste medium (SWM), which contains cellulose, lunar regolith flow-through, synthetic saliva, urea, starch, casamino acids, and lignin, supported reproducible fungal growth of 1.68 ± 0.37 g/L (in dry biomass), equivalent to 35.22% of the amount obtained under the nutrient-rich control medium (PDB). This SWM formulation distinctly outperformed the Potato Dextrose Broth control in absolute citric acid production by 10.78% (92.5 ± 23.3 mg/L vs. 83.5 ± 42.1 mg/L, respectively) and yielded a 301.20% increase (∼3-fold higher) in biomass-normalized specific citric acid yield (58.5 ± 26.8 mg/g vs. 19.4 ± 12.3 mg/g). This notable performance implies a fundamental shift in fungal carbon allocation away from primary growth, suggesting a biochemical pivot toward secondary metabolism. Our study reports a promising space waste based medium for fungal biotechnology to promote sustainable processes, both on Earth and in future long-term space missions.