On a future lunar habitat, acquiring needed resources in situ will inevitably come from the Lunar regolith. Biomining-the use of microorganisms to extract metals from the regolith-is sustainable and energy-efficient, making it highly promising for space exploration applications. Given the extensive use of filamentous fungi in industrial biotechnology, we investigated the ability of the fungus Penicillium simplicissimum to extract metals from the European Astronaut Centre lunar regolith simulant 1 (EAC-1A), which will be used as the analogue soil at the European Lunar Exploration Laboratory (LUNA) facility at the European Space Agency (ESA) and German Aerospace Centre (DLR) site. Biocompatibility tests demonstrated P. simplicissimum tolerance to high concentrations of EAC-1A regolith (up to 60 %), both on Earth gravity and Lunar simulated gravity. A fungal bioleaching setup was developed using a low nutrient medium, that allowed the fungus to extract metals from EAC-1A over the course of 2 weeks, including aluminium, iron, magnesium and calcium, among others. Metal recovery from the leachate achieved a promising average of 10 ± 3 g/L of metal powder. Our study demonstrates fungal biomining as a promising in situ resource utilization (ISRU) approach to be used in future missions to the Moon.